Expression of thrombin variants

ABSTRACT

One aspect of the invention contemplates a mutant E-WE thrombin precursor that contains the SEQ ID NO:1 amino acid residue sequence. Another aspect contemplates a thrombin precursor that contains the amino acid residue sequence Asp/Glu-Gly-Arg at positions 325, 326 and 327 based on the preprothrombin sequence. A third aspect contemplates a thrombin precursor that contains the SEQ ID NO:1 amino acid residue sequence as well as the amino acid residue sequence Asp/Glu Gly Arg at positions 325, 326 and 327 based on the preprothrombin sequence. Also contemplated is a composition that contains an effective amount of mutant thrombin dissolved or dispersed in a pharmaceutically acceptable carrier. A method is also disclosed for enhancing treating and preventing thrombosis in a mammal in need using that composition.

CROSS REFERENCE TO RELATED APPLICATIONS

The present invention claims priority to U.S. Provisional Patent Ser.No. 61/426,385, filed on Dec. 22, 2010, entitled Expression ofAnticoagulant Thrombin Mutants in Escherichia coli, whose disclosuresare incorporated herein by reference.

GOVERNMENTAL SUPPORT

The present invention was made with governmental support pursuant to thefollowing grants HL049413, HL058141, HL073813 and HL095315 awarded byNational Institutes of Health. The government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

Although substantial progress has been made in the prevention andtreatment of cardiovascular disease and its major risk factors, it hasbeen predicted that thrombotic complications will remain the leadingcause of death and disability and will represent a major burden toproductivity worldwide well into the year 2020 [Gerszten, (2008) Nature451:949-952]. Indeed, thrombosis is the most prevalent cause of fataldiseases in developed countries. It would be beneficial to have anantithrombotic agent that can be administered to patients with severeacute thrombotic diseases without the risk of causing hemorrhage, asexperienced with antithrombotic/thrombolytic therapy in the treatment ofacute ischemic stroke [Padma, (2005) Exp. Rev. Neurother. 5:223-233] orsystemic anticoagulants like heparin [Busch, (2004) Eur. J. Med. Res.9:199-206].

The gene for prothrombin encodes a protein referred to as preprothrombin[UniProtKB sequence P00734; SEQ ID NO:3] that contains 622 amino acidresidues composed of several distinct domains. The first domain is a 43amino acid residue leader sequence that is comprised of a signal peptidesequence and a propeptide. Cleavage of the leader sequence providesprothrombin that is comprised of Fragment-1 (residues 44-198),Fragment-2 (residues 199-327), and a fragment that contains the residuesof the thrombin A-chain (residues 328-363) and the thrombin B-chain(residues 364-622). The portion containing Fragment-2, and thrombin(residues 199-622) is referred to as prethrombin-1.

When expressed in vivo, prothrombin can be cleaved by the prothrombinasecomplex between residues Arg271 and Thr272 (henceforth, the prothrombinnumbering system is used unless otherwise specified) into a portioncontaining Fragment-1 and Fragment-2, and the prethrombin-2 fragment.Cleavage of prethrombin-2 between residues Arg320 and Ile321 and alsobetween residues Arg284 and Thr285 forms thrombin. This last cleavagecan be carried out by thrombin itself. Three letter code for amino acidresidues is usually used herein for the identification of single aminoacid residues. Single letter code is frequently used to identify two ormore residues such as the Ala-substituted Trp and Glu residues of WEthrombin [see, Cantwell, (2000) J. Biol. Chem. 275:39827-39830].Prothrombin can also be first cleaved between residues Arg320 and Ile321to form meizothrombin, and thereafter between residues Arg271 and Thr272and between residues Arg284 and Thr285 to form thrombin.

Alternatively, the enzyme ecarin can be used to cleave prothrombinbetween residues Arg271 and Thr272 to form meizothrombin. Autocatalyticprocessing results in the formation of meizothrombin desF1 and thenthrombin. Rhee et al. (1982) Biochemistry, 21:3437-3443.

Ecarin is a snake venom-derived protease isolated from Echis carinatus[Morita et al., (1978), J. Biochem. 83:559-570]. A cDNA encoding ecarinhas been cloned by Nishida et al., (1995) Biochemistry, 34:1771-1778].

Ecarin, a glycoprotein, is a metalloprotease, a mature form of which has426 amino acid residues in total, having a mosaic structure comprising aZn²⁺ chelate, a disintegrin domain and a Cys-rich domain. Ecarin cleavesproteins and peptides after the sequence Asp-Gly-Arg or Glu-Gly-Arg.Ecarin has been used to cleave prothrombin between residues Arg320 andIle321 to separate the A and B chains. U.S. Pat. No. 6,413,737.

Wild-type (wt) thrombin expressed from mammalian cells is often used forits procoagulant properties, particularly for the problem of surgicalbleeding. Thrombin variants engineered for optimal activity towardprotein C and minimal activity toward fibrinogen and protease-activatedreceptor 1 (PAR 1) have shown remarkable anticoagulant properties oftherapeutic interest both in vitro and in vivo [Cantwell, (2000) J.Biol. Chem. 275:39827-39830; Gibbs, (1995) Nature 378:413-416; Dang,Guinto, (1997) Nat. Biotechnol. 15:146-149; Gruber, (2002) J. Biol.Chem. 277:27581-27584; Gruber, (2006) J. Thromb. Haemost. 4:392-397;Gruber, (2007) Blood 109:3733-3740; Tsiang, (1996) Biochemstry35:16449-16457; Dang, (1997) J. Biol. Chem. 272:19649-19651; Griffin,(1195) Nature 378:337-338; Grinnell, (1997) Nat. Biotechnol. 15124-125].

The double mutant referred to as W215A/E217A (WE) is constructed bycombining the two single mutations W215A and E217A in the thrombinmolecule [Cantwell, (2000) J. Biol. Chem. 275:39827-39830]. W215A andE217A refer to amino acid residue positions in the thrombin amino acidresidue sequence using the position numbers as described in Bode et al.(1989) EMBO. J., 8:3467-3475, that correspond to sequential amino acidresidue positions 263 and 265 from the N-terminus of thrombin,respectively. A correlation table is provided hereinafter.

WE thrombin exhibits anticoagulant/antithrombotic activity both in vitroand in vivo [Arosio, (2000) Biochemistry 39:8095-8101; Cantwell, (2000)J. Biol. Chem. 275, 39827-39830; Berny, (2008) Arterioscler, Thromb.Vasc. Biol. 18:329-334; Feistritzer, (2006) J. Biol. Chem.281:20077-20084; Gruber, (2002) J. Biol. Chem. 277:27581-27584; Gruber,(2006) J. Thromb. Haemost. 4:392-397; Gruber, (2007) Blood 109,3733-3740]. Its antithrombotic effect in non-human primates is moreefficacious than the direct administration of activated protein C, andis safer to use than the administration of low molecular weight heparins[Gruber, (2007) Blood 109:3733-3740].

Activated protein C generated in situ with the mutant WE thrombin offerscytoprotective advantages over activated protein C administered to thecirculation [Feistritzer, (2006) J. Biol. Chem. 281:20077-20084].Furthermore, WE thrombin acts as a potent and safe antithrombotic byblocking the interaction of von Willebrand Factor with the plateletreceptor GpIb [Berny, (2008) Arterioscler, Thromb. Vasc. Biol.18:329-334; Gruber, (2007) Blood 109:3733-3740]. These properties of WEthrombin provide proof of principle that a thrombin mutant withpreferential activity toward protein C would be a compellinganticoagulant/antithrombotic agent in vivo. [Marino, (2010) J. Biol.Chem. 285:19145-19152].

Recombinant human coagulation enzymes, in particular WE, have beenexpressed and produced in animal cell cultures. Maintenance andpropagation of animal cell lines is complicated and expensive. Becausethrombin mutants for therapeutic use are being developed, the need hasemerged to produce these recombinant proteins in large quantities at anaffordable cost. Previously, thrombin was being produced in animalcells, namely baby hamster kidney cells. As animal cell cultures andlines can be expensive, an alternative is needed. The discussion belowillustrates an alternative expression technique that results inunexpected properties of the anticoagulant thrombin mutant WE.

BRIEF SUMMARY OF THE INVENTION

The present invention, in one aspect, contemplates a bacteria-derived(or -expressed) new recombinant E-WE thrombin enzyme precursor such asE-WE preprothrombin, E-WE prothrombin, E-WE prethrombin-1, E-WEprethrombin-2 and E-WE meizothrombin that contain the SEQ ID NO:1 aminoacid residue sequence and are preferably Escherichia coliculture-derived or -expressed (E. coli-derived; or E. coli-expressed). Acontemplated E-WE construct contains the SEQ ID NO:1 amino acid residuesequence, and preferably contains the SEQ ID NO: 5 amino acid residuesequence. A bacterially-expressed, glycosylation-free E-WE thrombin isalso contemplated that contains the SEQ ID NO:1 amino acid residuesequence.

It has unexpectedly been discovered that a recombinant E-WE thrombinprepared from a bacteria-expressed precursor is surprisingly safer andhas a greater anticoagulant (anti-thrombotic) therapeutic effect than aglycosylated WE thrombin expressed in a mammalian cell line from thesame DNA coding sequence.

In another aspect, the invention also contemplates a thrombin precursorvariant that can be activated by ecarin alone. Such a precursor variantpreferably has an amino acid residue sequence of up to 622 residues thatincludes the amino acid residue sequence Asp/Glu-Gly-Arg; i.e., one ofAsp or Glu peptide-bonded to Gly-Arg, (D/EGR), that is itselfpeptide-bonded to the N-terminal residue of the thrombin variant that isproduced; that is the incipient N-terminal residue of the thrombin Achain or the residue that becomes the new N-terminal residue of the Achain; i.e., Thr285.

Where the thrombin variant produced is human wild-type thrombin or aE-WE thrombin as disclosed herein, those D/EGR residues are preferablylocated right before Thr 285, at the positions 282, 283 and 284 of theconstruct (see, for example, SEQ ID NOs: 4, 5, 6, 7, 8, 16, 17, 20, 21and 22). This provides a wild-type or E-WE construct that can be cleaved(activated) using only ecarin. A most preferred E-WE precursor is theecarin-only cleavable prethrombin-2 polypeptide of SEQ ID NO:5.

Thus, one aspect of the invention contemplates a glycosylation-freerecombinant precursor of E-WE thrombin such as a E-WE preprothrombin, aE-WE meizothrombin, a E-WE prothrombin, a E-WE prethrombin-1, or a E-WEprethrombin-2 polypeptide that contains a tri-peptide, D/EGR, (ecarincleavage site) sequence at positions 326, 327 and 328 relative to theN-terminus of the preprothrombin polypeptide of SEQ ID NO: 4, orpositions 282, 283 and 284 of the prothrombin polypeptide SEQ ID NO: 19.A particularly preferred recombinant E-WE precursor such as apreprothrombin, E-WE meizothrombin, E-WE prothrombin and E-WEprethrombin-2 is expressed in Escherichia coli that is referred toherein as Escherichia coli-derived or -expressed (E. coli-derived or E.coli-expressed).

It should be understood that an E. coli-derived E-WE meizothrombin isformed by expression in E. coli followed by a post expression cleavagereaction that forms the A and B chains. However, E. coli-derived E-WEmeizothrombin is nonetheless referred to together with a single chainedprecursor such as E-WE preprothrombin, E-WE prothrombin, E-WEprethrombin-1, and E-WE prethrombin-2 for ease of expression.

It is also noted that a “zymogen” by many usual definitions is aninactive enzyme precursor that has to be acted upon to form the activeenzyme. Meizothrombins have some of the enzymatic activity of thrombin,but are also further acted upon to form thrombin. As such,meizothrombin, meizothrombin (des 1) an A-chain-shortened meizothrombin,and similar thrombin precursor compounds that have enzymatic activity,but must be acted upon to form thrombin are deemed active precursors ofthrombin herein.

The E-WE thrombin produced by activation of one of the above zymogens orproteolytic processing of active precursors is also particularlypreferred. It is believed that the enhanced antithrombotic activity andsafety observed for a contemplated E-WE thrombin results from thecompleteness of the cleavage reaction provided by the use of only ecarinthat permits a prepared E-WE thrombin to be substantially homogeneousand free from the presence of a N-terminally extended polypeptide in thethrombin A chain.

One contemplated bacteria-derived E-WE precursor includes the amino acidresidue sequence of SEQ ID NO:1. A contemplated E-WE thrombin containstwo chains, and also contains the amino acid residue sequence of SEQ IDNO:1. It is to be understood that E-WE thrombin contains a shorter (Achain) and longer (B chain) polypeptide linked together by a cystinebond, and that that sequence is read N-terminus to C-terminus as if thetwo polypeptides were one single chain, starting with the shorter Achain and followed by the longer B chain.

The terms “E-WE thrombin enzyme”, “recombinant E-WE thrombin enzyme”,and “E-WE thrombin” are often used herein as a short-hand phrase toencompass a contemplated glycosyl group-free E-WE thrombin enzyme havingthe human thrombin amino acid residue sequence in two chains andcontaining the B chain alanine for tryptophan (W) and alanine forglutamic acid (E) residue substitutions at positions 215 and 217 of thethrombin, respectively, using the position numbers as described in Bodeet al. (1989) EMBO. J., 8:3467-3475, that correspond to sequential aminoacid residue positions 263 and 265 from the N-terminus of thrombin,respectively.

A E-WE thrombin is a preferred polypeptide. Because a E-WE thrombinenzyme is a two-chain polypeptide and cannot be prepared from a singlepolypeptide chain without post expression processing, it is to beunderstood that a polynucleotide encoding each of the E-WE ecarinsite-containing precursors such as E-WE preprothrombin, E-WEprothrombin, E-WE meizothrombin, E-WE prethrombin-1, and E-WEprethrombin-2 polypeptides can be used to express a polypeptide that canitself be further processed with ecarin to provide a contemplated E-WEthrombin enzyme as an active agent for use in a contemplated compositionand/or method. In some aspects, a precursor that contains an ecarincleavage site peptide sequence bonded to the incipient thrombin A chainresidue can be expressed in an eukaryotic cell such as a mammalian cell,an insect cell, a plant cell or a yeast cell, or in a bacterial cellsuch as an E. coli cell.

Specifically contemplated is a E-WE precursor (such as E-WEpreprothrombin, E-WE prothrombin, E-WE meizothrombin, E-WEprethrombin-1, or E-WE prethrombin-2) mutant expressed in E. coli. Sucha specifically contemplated E-WE precursor includes the amino acidresidue sequence of SEQ ID NO:1, and preferably contains the amino acidresidue sequence of SEQ ID NO: 5 as discussed elsewhere herein.

Yet another aspect of the invention contemplates a pharmaceuticalcomposition that comprises an antithrombotic effective amount of abacteria-expressed recombinant E-WE thrombin dissolved or dispersed in apharmaceutically acceptable carrier. In one embodiment, a contemplatedcomposition is adapted to be administered parenterally. One suchcontemplated carrier is an isotonic aqueous buffer.

A contemplated composition is intended for therapeutic use for enhancinghemostasis or treating and preventing thrombosis. An illustrativetreatment comprises administering an above composition of anabove-described E. coli-derived recombinant E-WE thrombin enzyme to amammal in need thereof. It is contemplated that the administration isrepeated a plurality of times.

DEFINITIONS

The term “anticoagulant” as used herein refers to any agent or agentscapable of preventing or delaying blood clot formation in vitro and/orin vivo. The term “coagulation” as used herein refers to the process ofpolymerization of fibrin monomers, resulting in the transformation ofblood or plasma from a liquid to a gel phase. Coagulation of liquidblood can occur in vitro, intravascularly or at an exposed and injuredtissue surface. In vitro blood coagulation results in a gelled bloodthat maintains the cellular and other blood components in essentiallythe same relative proportions as found in non-coagulated blood, exceptfor a reduction in fibrinogen content and a corresponding increase infibrin. By “blood clot” is intended a viscous gel formed of, andcontaining all, components of blood in the same relative proportions asfound in liquid blood.

Thrombin is a serine endopeptidase (EC 3.4.21.5) that cleaves theArg-Gly bond in fibrinogen to form fibrin. Human thrombin is naturallymade in the body from a precursor polypeptide referred to herein aspreprothrombin that contains a single strand of 622 amino acid residues.Cleavage of that preprothrombin provides prothrombin, that contains asequence of C-terminal 579 amino acid residues (subject to potentialallelic variation or N-terminal microheterogeneity), plus the previousN-terminal pre-sequence of 43 residues that includes a signal peptide of24 residues at its N-terminus, and a propeptide of 19 residues bonded tothe C-terminus of the signal peptide [Degen et al. (1993) Biochemistry22:2087-2097].

Prothrombin is a zymogen, or inactive protease, that is activated by aseries of proteolytic cleavages to form thrombin. Prothrombin alsocontains the disulfide bond that is present in thrombin and links thetwo thrombin chains together. At least three sites in prothrombin arenormally subject to cleavage.

In vivo, prothrombin is cleaved between residues Arg271 and Thr272[residue numbers as described in Degen et al. (1993) Biochemistry22:2087-2097] (sequentially, preprothrombin Arg327-Thr328) bycoagulation Factor Xa (EC 3.4.21.6) another serine endopeptidase in thepresence of Factor Va, phospholipid and calcium ions to yieldprethrombin-2 and Fragment 1.2. The Fragment 1.2 polypeptide can also becleaved to form Fragment 1 and Fragment 2. The prethrombin-2 fragment iscleaved as discussed below to provide thrombin.

The prethrombin-2 polypeptide is the smallest naturally-occurringsingle-chain immediate precursor of thrombin (corresponding to residuesThr272 to Glu579 in prothrombin), has one glycosylation site at Asn373and four disulfide bonds, Cys293-Cys439, Cys348-Cys364, Cys493-Cys507,and Cys521-Cys551. The Cys293-Cys439 disulfide bond links the thrombin Achain (residues 272-320) and B chain (residues 321-579).

Prothrombin can also be proteolytically cleaved by the same enzymesystem between residues Arg320 and Ile321 (preprothrombin Arg363-Ile364)to yield meizothrombin, which in turn cleaves autolytically betweenArg155 and Ser156 (preprothrombin Arg198-Ser199) to produce Fragment 1(prothrombin 1-155; preprothrombin Ala43-Arg198) and meizothrombin des 1[a disulfide-linked dipeptide extending from original prothrombinresidue 156 (preprothrombin precursor Ser199) to the carboxy-terminus ofprothrombin].

Finally, thrombin is made from prethrombin-2 by further reaction withFactor Xa in the presence of Factor Va, phospholipid and calcium ions,this time to cleave between residues Arg320 and Ile321 (preprothrombinArg363-Ile364, or prothrombin, Arg328 and Ile329) and between residuesArg284 and Thr285. Thrombin can also be formed from meizothrombin des 1by proteolytic cleavage between Arg271 and Thr272 (prothrombin Arg271and Thr272). Cleavage between preprothrombin residues Arg363 and Ile364(prothrombin Arg320 and Ile321) forms the mature thrombin having adisulfide-bonded 36-residue light chain and 259-residue heavy chain.

The term “thrombin” as used herein refers to a multifunctional enzymethat contains up to about 300 residues in two polypeptide chainsconnected by a disulfide bond that exhibits at least two of theactivities exemplified in Table 3, hereinafter. Thrombin can act as aprocoagulant by the proteolytic cleavage of fibrinogen to fibrin.Thrombin can also activate the clotting Factors V (FV), VIII (FVIII), XI(FXI) and XIII (FXIII) leading to perpetuation of clotting, and thecleavage of the platelet thrombin receptor, PAR-1, leading to plateletactivation. Thrombin can also activate protein C.

Multiple antithrombotic mechanisms limit thrombin generation andactivity. When thrombin binds to thrombomodulin (TM), an integralmembrane protein on vascular endothelial cells, thrombin undergoes aconformational change and loses its procoagulant activity. Thrombin thenacquires the ability to convert the zymogen protein C (PC) to activatedprotein C (APC). APC, another serine endopeptidase (EC 3.4.21.69), actsas a potent anticoagulant by inactivating activated FV (FVa) and FVIII(FVIIIa), two essential cofactors in the clotting or coagulationcascade. APC also inactivates plasminogen activator inhibitor-1 (PAI-1),the major physiologic inhibitor of tissue plasminogen activator (tPA),thus potentiating normal fibrinolysis.

The term “coagulation cascade” as used herein refers to threeinterconnecting enzyme pathways as described, for example, by Manolin inWilson et al. (eds): Harrison's Principle of Internal Medicine,14.sup.th Ed. New York. McGraw-Mill, 1998, p. 341, incorporated hereinby reference in its entirety. The intrinsic coagulation pathway leads tothe formation of Factor IXa, that in conjunction with Factors VIIIa andX, phospholipid and Ca²⁺ provides Factor Xa. The extrinsic pathwayprovides Factor Xa and IXa after the combination of tissue factor andFactor VII. The common coagulation pathway interacts with the bloodcoagulation Factors V, VIII, IX and X to cleave prothrombin to thrombin(Factor IIa), which is then able to cleave fibrinogen to fibrin.

At least two distinct amino acid numbering systems are in use forthrombin in addition to the DNA-based system of Degen et al. [Degen etal. (1993) Biochemistry, 22:2087-2097.] One is based on alignment withchymotrypsinogen as described by Bode et al. and is the numbering systemused most widely in the protease field [Bode et al. (1989) EMBO. J.,8:3467-3475]. In a second system, the Sadler numbering scheme, the Bchain of thrombin commences with Ile1 and extends to Glu259, whereas theA chain is designated with “a” postscripts, as in Thr1a to Arg36a.

For example, Wu et al. have disclosed several thrombin mutants numberedin accordance with the Sadler scheme [Wu et al. (1991) Proc. Natl. Acad.Sci. U.S.A., 88:6775-6779). The Wu et al. mutants and the correspondingchymotrypsinogen and Degen et al. residue numbers, respectively, aresequentially shown as follows: His43 (57, 363), Lys52 (60f, 372), Asn53(60 g, 373), Arg62 (67, 382), Arg68 (73, 388), Arg70 (75, 390), Asp99(102, 419) and Ser205 (195, 525).

Throughout the present specification, the Bode et al. numbering systemis recited first to refer to amino acid residues for thrombin andthrombin mutants, and is often followed by a sequential numbering basedon the preprothrombin or prothrombin numbering. However, for thesequence listings corresponding to human recombinant thrombin enzymemutant W215A/E217A (E-WE; SEQ ID NO:1), and wild type (WT) humanthrombin (SEQ ID NO:2), a sequential numbering system is used. A thirdnumbering system based on the preprothrombin sequence of SEQ ID NO: 4 isalso sometimes used, particularly when a polypeptide longer thanthrombin is discussed.

Accordingly, amino acid positions 215 and 217 of thrombin and the E-WEthrombin enzyme as described in the present specification using the Bodeet al. system correspond to amino acid positions 263 and 265 of E-WEthrombin mutants and wild type thrombin in the sequential numberingsystem used in SEQ ID NOS:1 and 2. Those positions correspond toposition numbers 547 and 549 of the prothrombin sequence and to 590 and592 of the preprothrombin sequence of SEQ ID NOs:3 or 4.

A side-by-side comparison of the amino acid sequence for WT thrombin(prethrombin-2) using the Bode et al. sequential numbering system vs.the system used in SEQ ID NOS:1 and 2 is provided in Table A, below. Aslisted in Table A, the thrombin A-chain starts at amino acid residuenumber 1 of the sequential numbering system, whereas the thrombinB-chain starts at amino acid residue number 37.

TABLE A Sequential and Bode et al. Numbering forthe Amino Acid Residue Sequence of WildType Human Thrombin of SEQ ID NO: 2 Sequential Bode et al. Amino AcidPreprothrombin Number Number Residue Number   1    1h THR 328   2    1gPHE 329   3    1f GLY 330   4    1e SER 331   5    1d GLY 332   6    1cGLU 333   7    1b ALA 334   8    1a ASP 335   9   1 CYS 336  10   2 GLY337  11   3 LEU 338  12   4 ARG 339  13   5 PRO 340  14   6 LEU 341  15  7 PHE 342  16   8 GLU 343  17   9 LYS 344  18  10 LYS 345  19  11 SER346  20  12 LEU 347  21  13 GLU 348  22  14 ASP 349  23   14a LYS 350 24   14b THR 351  25   14c GLU 352  26   14d ARG 353  27   14e GLU 354 28   14f LEU 355  29   14g LEU 356  30   14h GLU 357  31   14i SER 358 32   14j TYR 359  33   14k ILE 360  34   14l ASP 361  35   14m GLY 362 36  15 ARG 363  37  16 ILE 364  38  17 VAL 365  39  18 GLU 366  40  19GLY 367  41  20 SER 368  42  21 ASP 369  43  22 ALA 370  44  23 GLU 371 45  24 ILE 372  46  25 GLY 373  47  26 MET 374  48  27 SER 375  49  28PRO 376  50  29 TRP 377  51  30 GLN 378  52  31 VAL 379  53  32 MET 380 54  33 LEU 381  55  34 PHE 382  56  35 ARG 383  57  36 LYS 384  58  36a SER 385  59  37 PRO 386  60  38 GLN 387  61  39 GLU 388  62  40LEU 389  63  41 LEU 390  64  42 CYS 391  65  43 GLY 392  66  44 ALA 393 67  45 SER 394  68  46 LEU 395  69  47 ILE 396  70  48 SER 397  71  49ASP 398  72  50 ARG 399  73  51 TRP 400  74  52 VAL 401  75  53 LEU 402 76  54 THR 403  77  55 ALA 404  78  56 ALA 405  79  57 HIS 4406  80  58CYS 407  81  59 LEU 408  82  60 LEU 409  83   60a TYR 410  84   60b PRO411  85   60c PRO 412  86   60d TRP 413  87   60e ASP 414  88   60f LYS415  89   60g ASN 416  90   60h PHE 417  91   60i THR 418  92  61 GLU419  93  62 ASN 420  94  63 ASP 421  95  64 LEU 422  96  65 LEU 423  97 66 VAL 424  98  67 ARG 425  99  68 ILE 426 100  69 GLY 427 101  70 LYS428 102  71 HIS 429 103  72 SER 430 104  73 ARG 431 105  74 THR 432 106 75 ARG 433 107  76 TYR 434 108  77 GLU 435 109   77a ARG 436 110  78ASN 437 111  79 ILE 438 112  80 GLU 439 113  81 LYS 440 114  82 ILE 441115  83 SER 442 116  84 MET 443 117  85 LEU 444 118  86 GLU 445 119  87LYS 446 120  88 ILE 447 121  89 TYR 448 122  90 ILE 449 123  91 HIS 450124  92 PRO 451 125  93 ARG 452 126  94 TYR 453 127  95 ASN 454 128  96TRP 455 129  97 ARG 456 130   97a GLU 457 131  98 ASN 458 132  99 LEU459 133 100 ASP 460 134 101 ARG 461 135 102 ASP 462 136 103 ILE 463 137104 ALA 464 138 105 LEU 465 139 106 MET 466 140 107 LYS 467 141 108 LEU468 142 109 LYS 469 143 110 LYS 470 144 111 PRO 471 145 112 VAL 472 146113 ALA 473 147 114 PHE 474 148 115 SER 475 149 116 ASP 476 150 117 TYR477 151 118 ILE 478 152 119 HIS 479 153 120 PRO 480 154 121 VAL 481 155122 CYS 482 156 123 LEU 483 157 124 PRO 484 158 125 ASP 485 159 126 ARG486 160 127 GLU 487 161 128 THR 488 162 129 ALA 489 163  129a ALA 490164  129b SER 491 165  129c LEU 492 166 130 LEU 493 167 131 GLN 494 168132 ALA 495 169 133 GLY 496 170 134 TYR 497 171 135 LYS 498 172 136 GLY499 173 137 ARG 500 174 138 VAL 501 175 139 THR 502 176 140 GLY 503 177141 TRP 504 178 142 GLY 505 179 143 ASN 506 180 144 LEU 507 181 145 LYS508 182 146 GLU 509 183 147 THR 510 184 148 TRP 511 185 149 THR 512 186 149a ALA 513 187  149b ASN 514 188  149c VAL 515 189  149d GLY 516 190 149e LYS 517 191 150 GLY 518 192 151 GLN 519 193 152 PRO 520 194 153SER 521 195 154 VAL 522 196 155 LEU 523 197 156 GLN 524 198 157 VAL 525199 158 VAL 526 200 159 ASN 527 201 160 LEU 528 202 161 PRO 529 203 162ILE 530 204 163 VAL 531 205 164 GLU 532 206 165 ARG 533 207 166 PRO 534208 167 VAL 535 209 168 CYS 536 210 169 LYS 537 211 170 ASP 538 212 171SER 539 213 172 THR 540 214 173 ARG 541 215 174 ILE 542 216 175 ARG 543217 176 ILE 544 218 177 THR 545 219 178 ASP 546 220 179 ASN 547 221 180MET 548 222 181 PHE 549 223 182 CYS 550 224 183 ALA 551 225 184 GLY 552226  184a TYR 553 227 185 LYS 554 228 186 PRO 555 229  186a ASP 556 230 186b GLU 557 231  186c GLY 558 232  186d LYS 559 233 187 ARG 560 234188 GLY 561 235 189 ASP 562 236 190 ALA 563 237 191 CYS 564 238 192 GLU565 239 193 GLY 566 240  94 ASP 567 241 195 SER 568 242 196 GLY 569 243197 GLY 570 244 198 PRO 571 245 199 PHE 572 246 200 VAL 573 247 201 MET574 248 202 LYS 575 249 203 SER 576 250 204 PRO 577 251  204a PHE 578252  204b ASN 579 253 205 ASN 580 254 206 ARG 581 255 207 TRP 582 256208 TYR 583 257 209 GLN 584 258 210 MET 585 259 211 GLY 586 260 212 ILE587 261 213 VAL 588 262 214 SER 589 263 215 TRP 590 264 216 GLY 591 265217 GLU 592 266 219 GLY 593 267 220 CYS 594 268 221 ASP 595 269  221aARG 596 270 222 ASP 597 271 223 GLY 598 272 224 LYS 599 273 225 TYR 600274 226 GLY 601 275 227 PHE 602 276 228 TYR 603 277 229 THR 604 278 230HIS 605 279 231 VAL 606 280 232 PHE 607 281 233 ARG 608 282 234 LEU 609283 235 LYS 610 284 236 LYS 611 285 237 TRP 612 286 238 ILE 613 287 239GLN 614 288 240 LYS 615 289 241 VAL 616 290 242 ILE 617 291 243 ASP 618292 244 GLN 619 293 245 PHE 620 294 246 GLY 621 295 247 GLU 622

A contemplated E-WE precursor and E-WE thrombin has sequence identity tothe amino acid residue sequence of a human thrombin that has alanineamino acid residue substitutions at residue positions 215 and 217 ofthrombin of SEQ ID NO:2, as determined by sequence alignment programsand parameters described elsewhere herein. The thrombin portion of acontemplated recombinant E-WE thrombin precursor such as E-WEpreprothrombin, E-WE prothrombin, E-WE meizothrombin, E-WEprethrombin-1, E-WE prethrombin-2 or E-WE thrombin has the amino acidresidue sequence of SEQ ID NO:1, treating E-WE meizothrombin and E-WEthrombin as if they were each single chained molecules.

In one embodiment of the present invention, a E-WE preprothrombin,prothrombin, meizothrombin, E-WE prethrombin-1, prethrombin-2 orthrombin has both an active (catalytic) site and exosite I available forbinding to direct thrombin inhibitors (DTIs). The active site cleft ofthrombin is bordered by two prominent insertion loops (i.e., the 60-loopand the 148-loop) that control, in part, the interaction of substratesand inhibitors with the active site [Bode et al. (1989) EMBO J.,8:3467-3475; Le Bonniec et al. (1993) J. Biol. Chem., 268:19055-19061;Le Bonniec et al. (1992) J. Biol. Chem., 267:19341-19348].

Exosites I and II are electropositive sites in near-opposition on thesurface of thrombin known to bind to a number of substrates (Stubbs andBode (1993) Thromb. Res., 69:1-58; Bode et al. (1992) Protein Sci.,1:26-471). For example, exosite I is known to bind fibrinogen and fibrinI and II (see, for example, Naski et al. (1990) J. Biol. Chem.,265:13484-13489; Naski and Shafer (1991) J. Biol. Chem.,266:13003-13010), whereas exosite II is known to bind heparin and otherglycosaminoglycans (Bode et al. (1992) Protein Sci., 1:26-471; Gan etal. (1994) J. Biol. Chem., 269:1301-1305).

The term “procoagulant” as used herein refers to agents that initiate oraccelerate the process of blood coagulation through the transformationof soluble circulating fibrinogen to an insoluble cross-linked, fibrinnetwork. An exemplary procoagulant is native thrombin thatproteolytically cleaves fibrinogen to fibrin. In vitro, the procoagulantultimately yields a blood clot. In vivo, a procoagulant typically yieldsa thrombus under pathological conditions.

The term “thrombus” as used herein refers to a coagulated intravascularmass formed from the components of blood that results from apathological condition of an animal or human. Typically, theconstituents of a thrombus have relative proportions differing fromthose of the same components in circulating blood. A thrombus isgenerated in vivo by a dynamic process that comprises cleavage offibrinogen to fibrin, activation of platelets and the adherence ofplatelets to the cross-linked fibrin network.

“Reduced procoagulant” or “anticoagulant” or “antithrombotic” activity,as used herein, can be determined for a E-WE thrombin through thecalculation of its PA/FC ratio (also called “relative anticoagulantpotency” or “RAP”) [see, e.g., Di Cera (1998) Trends Cardiovasc. Med.,8:340-350; Dang et al. (1997) Nat. Biotechnol., 15:146-149]. The term“PA/FC ratio” refers to the ratio of the percent of wild-type protein Cactivation (PA) activity exhibited by a thrombin relative to the percentof wild-type fibrinogen clotting (FC) activity of that thrombin. A valueof PA/FC greater than 1.0 indicates that the E-WE thrombin has reducedprocoagulant fibrinogen cleavage activity relative to the residualantithrombotic activity resulting from protein C activation.

The present invention has several benefits and advantages.

One benefit is that the costly and time-consuming activation processrequiring the use of Factor Xa in the presence of Factor Va,phospholipid and calcium ions is not required.

An advantage of the invention is that E-WE thrombin formation from aE-WE precursor that contains the 4 mutations relative to wt thrombin canbe carried out in a single reaction using ecarin.

Another benefit of the invention is that the E-WE thrombin prepared froma contemplated E-WE precursor exhibits greater safety and antithromboticactivity than the glycosylated E-WE thrombin produced in a mammaliancell culture, whereas other thrombins produced in mammalian andbacterial cells had similar activities.

Another advantage of the present invention is that preparation of a E-WEprecursor in bacterial culture lowers the possible risks ofcontamination with a mammalian pathogen or allergen.

A yet further benefit of the invention is that the production of E-WEthrombin is less costly using a contemplated E-WE precursor as areactant and expression from bacteria instead of mammalian cells.

A still further benefit of the invention is that introduction of ecarincleavage sites into the mutant E-WE thrombin precursorW215A/E217A/N282D/P283G that replaces the usual prothrombinase cleavagesite located up-stream of and immediately adjacent to the incipientN-terminal residue of a mature thrombin molecule provides a newmolecular entity and permits formation of the A chain N-terminal residueand the A and B chain cleavage using the same enzyme, which overcomes orminimizes the greatly reduced rate of Factor Xa-catalyzed cleavage of aE-WE precursor at that position.

A still further benefit of the invention is that introduction of anecarin cleavage site into a precursor that replaces the Factor Xa siteat prothrombin position 184 provides a new molecular entity and permitsformation of the A chain N-terminal residue and the A and B chaincleavage using the same enzyme, which overcomes or minimizes the greatlyreduced rate of Factor Xa-catalyzed cleavage of a E-WE precursor at thatposition.

Still further benefits and advantages will be apparent to a worker ofordinary skill from the detailed description that follows.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the present invention contemplates a bacteria-derived(-grown or -expressed) recombinant E-WE thrombin precursor such as E-WEpreprothrombin, E-WE prothrombin, E-WE prethrombin-1, E-WE prethrombin-2or E-WE meizothrombin that contains the SEQ ID NO:1 amino acid residuesequence and are preferably Escherichia coli culture-derived (or-expressed; E. coli-derived; E. coli-expressed). A contemplated E-WEprecursor more preferably contains the SEQ ID NO: 5 amino acid residuesequence. A bacterially-expressed, glycosylation-free E-WE thrombin isalso contemplated that contains the SEQ ID NO:1 amino acid residuesequence.

It has unexpectedly been discovered that an E. coli-derived recombinantE-WE thrombin enzyme is safer and has a greater antithrombotictherapeutic effect than its mammalian cell-expressed (-derived) analogueproduced from the same DNA coding sequence. Therefore, abacteria-derived E-WE thrombin enzyme is more useful than mammalian cellline-derived E-WE thrombin for the treatment and prevention of diseasesthat are associated with pathological blood coagulation.

A particular feature of bacterially-expressed polypeptides and proteinsis that those expression products are non-glycosylated or glycosyl-freepolypeptides and proteins. As a consequence, a contemplated tobacteria-derived (-expressed) E-WE thrombin or E-WE thrombin precursorcan also be referred to as a non-glycosylated or glycosyl-free E-WEthrombin or E-WE thrombin precursor.

The present invention provides a recombinant E-WE thrombin enzyme, acomposition and a method for inhibiting the effect of coagulants invitro as well as in vivo. As discussed herein, E-WE precursors such asrecombinant E-WE preprothrombin, E-WE prothrombin, E-WE meizothrombin,E-WE prethrombin-1, E-WE prethrombin-2, and E-WE thrombin arecontemplated in which an alanine residue replaces each of a tryptophanand a glutamic acid residue at positions 215 and 217, respectively, inthe thrombin sequence that is referred to as W215A/E217A or E-WE. Theseenzymes can be used in various applications to improve therapeuticefficacy and safety.

Also contemplated are a precursor that contains an ecarin cleavage sitecomprising the sequence Asp/Glu-Gly-Arg peptide-bonded to the incipientN-terminal residue of the thrombin A chain, that is, at residuepositions 325-327 relative to the N-terminus of preprothrombin of SEQ IDNO:6. A contemplated thrombin precursor polypeptide preferably has anamino acid residue sequence of up to about 622 residues as inpreprothrombin. It is preferred that the Asp/Glu-Gly-Arg sequencecleavage site be present 298-296 amino acid residues from thecarboxy-terminus.

Four and five residue ecarin cleavage sites are also contemplated.Illustrative sites include the sequences Asp/Glu-Gly-Arg;Ile-Asp/Glu-Gly-Arg; Asp/Glu-Gly-Arg-Ile; Ile-Asp/Glu-Gly-Arg-Ile-Val(SEQ ID NO: 14); and Asp/Glu-Gly-Arg-Ile-Val-Glu (SEQ ID NO: 15), wherein each case, the ecarin cuts after the Arg residue of the site.

In another aspect, a contemplated precursor polypeptide, except for theAsp/Glu-Gly-Arg sequence, contains the amino acid residue sequence ofwild type human thrombin of SEQ ID NO:2. An illustrative precursor isthat of sequence of SEQ ID NOs:6 or 7.

In another aspect of the invention, a precursor polypeptide containingan Asp/Glu-Gly-Arg sequence located as discussed above, is part of anamino acid residue sequence whose thrombin portion (the portion thatforms thrombin) is at least about 95 percent identical to the amino acidresidue sequence of wild type human thrombin of SEQ ID NO:2. Morepreferably, a thrombin portion is about 97 percent or more identical tothat of wild type human thrombin of SEQ ID NO:2, most preferably, theidentity is about 98 percent or more.

Illustrative non-wild type ecarin-cleavable thrombin precursors arethose of sequence of SEQ ID NOs:4, 5, 8, 16 and 20. Another group ofillustrative non-wild type thrombin precursors include the amino acidsequence of SEQ ID NOs:1, 9 or 10.

Inasmuch as thrombin contains 295 amino acid residues, a contemplatedthrombin portion can differ from wild type human thrombin of SEQ ID NO:2by up to about 15 residues. A more preferred thrombin portion of athrombin precursor can differ from wild type human thrombin of SEQ IDNO:2 by about nine amino acid residues. Illustratively, a thrombinprecursor containing the Δ146-149e deletion of thrombin sequentialpositions 182-188 is missing seven thrombin residues and thereforediffers from the thrombin of SEQ ID NO:2 by seven residues. A thrombinprecursor containing sequence a W215G mutation along with the Δ146-149edeletion (W215G/A146-149e) differs from wild type human thrombin of SEQID NO:2 by eight amino acid residues, as does W215E/Δ146-149e. A E-WEthrombin precursor also containing a Δ146-149e deletion differs by nineresidues.

In addition to thrombin and thrombin precursors containing the E-WEsubstitutions, the present invention provides a recombinant E-WEthrombin precursor (E-WE preprothrombin, E-WE prothrombin, E-WEmeizothrombin, E-WE prethrombin-2) that also contains an added ecarincleavage site at positions 325, 326 and 327 from the N-terminus ofpreprothrombin, where the site is Asp/Glu-Gly-Arg. A contemplated E-WEthrombin precursor has the sequence of SEQ ID NO: 4, and more preferablyof the ecarin-cleavable pethrombin-2 polypeptide of SEQ ID NO:5.

It is also to be noted that a contemplated thrombin precursor need notbe a well known thrombin precursor as discussed above. Rather, acontemplated thrombin precursor can be viewed as a expressible fusionprotein (polypeptide) in which the N-terminal portion of the fusionpolypeptide provides a convenient sequence for expression and/orpurification (expression/purification), whose C-terminal residue ispeptide-bonded to an ecarin cleavage sequence as discussed above, whoseArg residue is peptide-bonded to the carboxy-terminal portion that isthe thrombin sequence desired to be expressed. Thus, the N-terminalportion of the expressed fusion polypeptide (protein) is a convenientexpression/purification sequence, whereas the C-terminal portion has adesired thrombin sequence, and the two portions are joined (linked) bythe amino acid residue sequence of an ecarin cleavage site.

Thus, an exemplary N-terminal fusion polypeptide portion can be acommonly expressed polypeptide such as FLAG peptide, β-galactosidase(β-Gal or LacZ), glutathione-S-transferase (GST) protein, a hexa-hispeptide (6×His-tag), chitin binding protein (CBP), maltose bindingprotein (MBP), V5-tag, c-myc-tag, HA-tag, and the like as are wellknown. The carboxy-terminus of the N-terminal fusion polypeptide portionis peptide bonded to an ecarin cleavage site as discussed above and theArg of that ecarin cleavage sequence is peptide-bonded to the incipientN-terminal residue of a desired thrombin sequence that constitutes thecarboxy-terminal portion of the fusion protein or polypeptide.

An illustrative desired thrombin sequence is that of wild type humanthrombin of SEQ ID NO:2, E-WE thrombin of SEQ ID NO:1, Δ146-149ethrombin and the like. Illustrative carboxy-terminal thrombin portionsof such fusion polypeptides include the ecarin-activatable E-WE thrombinprecursor A of SEQ ID NO:16 and the ecarin-activatable thrombinprecursor A of SEQ ID NO:17. Further examples of sequences of acarboxy-terminal thrombin portion and the linking ecarin cleavage sitefor a E-WE thrombin and for a wild-type thrombin are illustrated by SEQID NOs: 21 and 22.

The present invention enables large scale production of a recombinantthrombin, a E-WE thrombin precursor, such as E-WE preprothrombin, E-WEprothrombin, E-WE meizothrombin, E-WE prethrombin-2 and thrombin enzymeW215A/E217A (E-WE) for in vitro and in vivo studies, therapies and otherapplications that are discussed herein. A contemplated bacterialexpression product also preferably contains the ecarin cleavage sitepresent in the SEQ ID NO:5 amino acid residue sequence. In addition,this type of large scale production is cost-effective compared tocommonly used thrombin, meizothrombin, prethrombin-2 or prothrombinexpression in baby hamster kidney (BHK) or other mammalian cells.

The invention also provides a form of E-WE thrombin enzyme that issignificantly less active toward the procoagulant substrate fibrinogenthan the BHK-expressed version. Moreover, a recombinant E-WEpreprothrombin, E-WE prothrombin, E-WE meizothrombin, or E-WEprethrombin-2 expressed in E. coli provides a more potent anti-coagulantE-WE thrombin than the corresponding E-WE thrombin expressed in BHKcells, and therefore permits use of lower effective doses when used forthe treatment of disease.

Wild type (WT) human thrombin and other anticoagulant mutants studiedexpressed in E. coli are not more potent than their BHK-expressedcounterparts, but rather, have the same potency. Accordingly, an E.coli-derived recombinant E-WE thrombin enzyme and a E-WE thrombinprecursor such as a E-WE meizothrombin, a E-WE prethrombin-2, a E-WEprothrombin, and a E-WE preprothrombin from which E-WE thrombin can beprepared, can be more useful therapeutically or in other uses than a BHKcell-derived E-WE thrombin, E-WE prethrombin-2, E-WE prothrombin, orE-WE preprothrombin expressed from the same coding DNA sequence.

The reason for the enhanced activity of a contemplated E. coli-derivedrecombinant E-WE thrombin enzyme compared to the same precursor beingexpressed in a mammalian cell is not known with certainty. However, thatactivity difference is believed to be due to imperfect cleavage of themammalian-expressed precursor WE prethrombin-2, WE prothrombin or thelike in which some of the up-stream residues from the thrombin A chainN-terminus remain bonded after the activation (cleavage) step. It ispresumed that the replacement of the Trp and Glu residues with Alaresidues in a contemplated E-WE thrombin causes some interference withpost expression processing after E. coli expression that is not presentin wild type thrombin or other mammalian-expressed thrombin mutantsstudied. This presumption is underscored by the fact that E-WE thrombinhas a greatly reduced rate of proteolytic catalysis at the Arg284auto-proteolytic site that reduces the length of the A chain by thirteenamino acid after the Factor Xa cleavage at Arg271. It is postulated thatthese additional residues on the mammalian-expressed WE construct, thatare eliminated by introduction of the ecarin site at the 282-284positions, reduce the anticoagulant potency of mammalian-expressed WE.

As can be seen from the data that follow in Table 3, several separatepreparations of a contemplated E. coli-derived recombinant E-WE thrombinprovided similar activity results that are within usual activity assayvariances. The E-WE thrombin prepared using only ecarin exhibitedsimilar activities that also were different from the activity of WEthrombin expressed in BHK cells.

A contemplated E-WE thrombin expressed in bacteria (e.g., E. coli) isfree of glycosylation and can be used therapeutically, such as forenhancing hemostasis or treating and preventing thrombosis.

One advantage of the present invention is that it permits faster andmore economical production of large quantities of anticoagulant(antithrombotic) thrombin. In particular, bacteria such as E. coli canbe used to produce large batches of a E-WE thrombin enzyme forpharmaceutical development, therapy and other uses.

Compositions and Methods

A pharmaceutical composition or formulation is also contemplated thatcontains an effective amount of a contemplated bacteria-expressed E-WEthrombin enzyme dissolved or dispersed in a pharmaceutically acceptablecarrier.

The pharmaceutical composition can be used for treating variousdiseases. For example, systemic and local use of the pharmaceuticalcomposition of the present invention can be used for enhancingantithrombotic activity in a mammal at risk of or having intravascularblood coagulation. Another use for the pharmaceutical composition is totreat thrombotic diseases of one or more organs.

A contemplated E. coli-derived recombinant E-WE thrombin is dissolved ordispersed in a composition that is pharmaceutically acceptable andcompatible with the active ingredient as is well known. The phrases“pharmaceutically acceptable” or “physiologically tolerable” refer tomolecular entities and compositions that typically do not produce anallergic or similar untoward reaction, and the like, when administeredto a host mammal.

The amount of E. coli-derived recombinant E-WE thrombin utilized in eachadministration is referred to as an antithrombotic effective amount andcan vary widely, depending inter alia, upon the genus of the mammal towhich a composition is administered, and the severity of the diseasestate being treated. An effective amount of an E. coli-derivedrecombinant E-WE thrombin enzyme at least temporarily improves thedisease state for which the protein is administered.

A contemplated pharmaceutical composition for parenteral use comprisesan effective amount of E. coli-derived antithrombotic E-WE thrombindissolved or dispersed in a pharmaceutically acceptable carrier. Auseful pharmaceutically acceptable carrier for parenteral administrationis typically aqueous and is a liquid at room temperature.

An effective amount of a contemplated E. coli-derived antithromboticE-WE thrombin administered per day is typically based on the weight ofthe recipient in kilograms. In one preferred embodiment, a dose of apharmaceutical composition contains an antithrombotic effective amountof about 0.1 μg/kg/day to about 1 mg/kg/day of an E. coli-derivedanticoagulant E-WE thrombin.

The term parenteral as used herein includes subcutaneous injections,intravenous, intramuscular, intrasternal injection, or infusiontechniques. Formulation of drugs is discussed in, for example, Hoover,John E., Remington's Pharmaceutical Sciences, Mack Publishing Co.,Easton, Pa.; 1975 and Liberman, H. A. and Lachman, L., Eds.,Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980.

Injectable preparations, for example, are typically sterile injectableaqueous preparations, aqueous/alcoholic preparations, and can alsoinclude oleaginous suspensions that can be formulated according to theknown art using suitable dispersing or wetting agents and suspendingagents. A sterile injectable preparation can also be a sterileinjectable solution or suspension in a nontoxic pharmaceuticallyacceptable diluent or solvent, for example, as a solution in ethanol,1,3-butanediol or propylene glycol.

More specifically, among the pharmaceutically acceptable vehicles andsolvents that can be employed are water, Ringer's solution, isotonicsodium chloride solution, and phosphate-buffered saline. Liquidparenteral compositions also include, for example, sterile watersolutions of an active component or sterile solution of the activecomponent in solvents comprising water, ethanol, or propylene glycol.

In addition, sterile, fixed oils are conventionally employed as asolvent or suspending medium. For this purpose any bland fixed oil canbe employed including synthetic mono- or diglycerides. In addition,fatty acids such as oleic acid find use in the preparation ofinjectables. Dimethyl acetamide, surfactants including ionic andnon-ionic detergents, polyethylene glycols can be used. Mixtures ofsolvents and wetting agents such as those discussed above are alsouseful.

Sterile solutions can be prepared by dissolving the active component inthe desired solvent system, and then passing the resulting solutionthrough a membrane filter to sterilize it or, alternatively, bydissolving or suspending the sterile compound in a previously sterilizedsolvent under sterile conditions. A contemplated composition can also besterilized by passage through a beam of ionizing radiation. The use ofconventional preservatives and antibacterial agents are alsocontemplated.

Suitable carriers for parenteral use can take a wide variety of formsdepending on the intended use and are, for example, aqueous solutionscontaining saline, phosphate buffered saline (PBS), dextrose, glycerol,ethanol, or the like and combinations thereof. In addition, if desired,a composition can contain minor amounts of auxiliary pharmaceuticallysuitable substances such as wetting or emulsifying agents,preservatives, acids, bases, salts, sugars, pH buffering agents, whichenhance the effectiveness of the composition.

Typical dosage for a contemplated anti-coagulant E. coli-expressed E-WEthrombin composition for systemic treatment of pathological coagulation(thrombosis) is about 0.1 μg/kg/hour to about 1 mg/kg/hour. A dose of atopical composition of E. coli-expressed procoagulant thrombin cancontain about 10 μg to about 10 mg of enzyme.

A contemplated topical composition of E. coli expressed procoagulantthrombin analogs, including wt thrombin can use as its base a solution,spray, cream, a gel, medicated strips, or another acceptable base todeliver an effective amount of E. coli-derived procoagulant thrombinenzyme to the wound. Components of compositions for topical application(administration) are well known in the art and can be found in manytexts such as Hoover, John E., Remington's Pharmaceutical Sciences, MackPublishing Co., Easton, Pa.; 1975, or a later edition thereof.

Thus, for example, an external or topical preparation for application toa wound, incision or other break in the skin is contemplated. Thiscomposition contains an effective amount of an ecarin-activatedprocoagulant wild-type thrombin dissolved or dispersed in a gel basethat also comprises about 0.5 to about 50% by weight of a water-solublehigh molecular weight gellant (gelling agent). A contemplated gel baseincludes about 30 to about 70% by weight water, and zero to 70% byweight of a water-retaining agent. Further ingredients such ascolorants, preservatives and other well known excipients can also bepresent. The procoagulant wild-type thrombin can also be used as acomponent of fibrin sealants, to be used topically, for bleedingcontrol.

Illustrative gellants include naturally occurring gelling agents such asgelatin, starch, agar, mannan, alginic acid, gum arabic, gum tragacanth,karaya gum and locust bean gum, as well as synthetic gelling agents suchas cross-linked polyacrylic acid, a salt of cross-linked polyacrylicacid, and copolymers thereof, dextrin, methylcellulose, methylcellulosesodium, carboxymethylcellulose, carboxymethylcellulose sodium, polyvinylalcohol, partially hydrolyzed polyvinyl acetate, polyvinyl pyrrolidone,a copolymer of methyl vinyl ether and maleic anhydride. Illustrativewater-retaining agents include ethylene glycol, diethylene glycol,polyethylene glycol, glycerin, sorbitol, martitol, propylene glycol and1,3-butylene glycol.

A mammal in need of treatment and to which a pharmaceutical compositioncontaining a contemplated E. coli-expressed anticoagulant E-WE thrombinis administered can be a primate including a human, an ape such as achimpanzee or gorilla, a monkey such as a cynomolgus monkey or amacaque. The treated mammal also includes a laboratory animal such as arat, mouse or rabbit, a companion animal such as a dog, cat, horse, or afood animal such as a cow or steer, sheep, lamb, pig, goat, llama or thelike.

A contemplated composition can be administered to a mammal in need as isnecessary to achieve the degree of anticoagulant activity desired. TheE-WE thrombin molecule present in such a composition can be formed insitu (in vivo or in vitro) using activating enzymes and other well knowncofactors present at the site of administration or within the body ofthe mammal. The E-WE thrombin can also be obtained prior toadministration by admixture of i) a pharmaceutical compositioncontaining a thrombin precursor such as E-WE preprothrombin, E-WEprothrombin or E-WE prethrobmin-2 and ii) the well known activatingenzymes and cofactors as is illustrated hereinafter. More preferably,the E-WE thrombin is prepared from a E-WE thrombin precursor containingthe amino acid residue sequence of SEQ ID NO:5 using ecarin to form theE-WE thrombin. Illustratively, a pharmaceutical composition containing aE-WE thrombin precursor fusion polypeptide can be contacted withecarin-bound Sepharose® particles in an activation column that alsocontains antibody combining sites that bind to the cleaved N-terminalportion of the precursor prior to being administered to the mammal.

Methods for making the proteins and nucleotides used in the invention,as well as the methods of the invention taught in this disclosureutilize the conventional techniques of molecular genetics, cell biology,and biochemistry. Useful methods in molecular genetics, cell biology andbiochemistry are described in Molecular Cloning: A Laboratory Manual,2nd Ed. (Sambrook et al., 1989); Oligonucleotide Synthesis (M. J. Gait,ed., 1984); Animal Cell Culture (R. I. Freshney, ed., 1987); the seriesMethods in Enzymology (Academic Press, Inc.); “Gene Transfer Vectors forMammalian Cells” (J. M. Miller & M. P. Calos, eds., 1987); CurrentProtocols in Molecular Biology and Short Protocols in Molecular Biology,3rd Edition (F. M. Ausubel et al., eds., 1987 & 1995); and RecombinantDNA Methodology II (R. Wu ed., Academic Press 1995). Methods for peptidesynthesis and manipulation are described in Solid Phase PeptideSynthesis, (J. M. Stewart & J. D. Young, 1984); Solid Phase PeptideSynthesis: A Practical Approach (E. Atherton & R. C. Sheppard, 1989);The Chemical Synthesis of Peptides (J. Jones, International Series ofMonographs on Chemistry vol. 23, 1991); and Solid Phase PeptideSynthesis, (G. Barany & R. B. Merrifield, Chapter 1 of The Peptides,1979); and Bioconjugate Techniques (G. T. Hermanson, 1996).

In some embodiments, a contemplated thrombin precursor is expressed ineukaryotic host cells. The thrombin precursor polypeptide so expressedis glycosylated. Illustrative eukaryotic cells include insect cells suchas Sf9, and mammalian cell lines such as CHO, COS, 293, 293-EBNA, BHK,HeLa, NIH/3T3, and the like. Exemplary yeast host cells includeSaccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha,Kluyveromyces lactis, Schwanniomyces occidentis, Schizosaccharomycespombe and Yarrowia lipolytica.

More preferably, a contemplated thrombin precursor polypeptide isexpressed in prokaryotic cells. Preferred prokaryotic cells are bacteriacells. Preferred bacteria cells are E. coli cells. Several strains ofSalmonella such as S. typhi and S. typhimurium and S. typhimurium-E.coli hybrids can also be used to express a contemplated thrombinprecursor. See, U.S. Pat. No. 6,024,961; U.S. Pat. No. 5,888,799; U.S.Pat. No. 5,387,744; U.S. Pat. No. 5,297,441; Ulrich et al., (1998) Adv.Virus Res., 50:141-182; Tacket et al., (1997) Infect. Immun.,65(8):3381-3385; Schödel et al., (1997) Behring Inst. Mitt., 98:114-119;Nardelli-Haefliger et al., (1996) Infect. Immun., 64(12):5219-5224;Londono et al., (1996) Vaccine, 14(6):545-552, and the citationstherein.

A preferred E. coli strain useful herein for expression of acontemplated E-WE thrombin enzyme is BL21 (DE3). Additional E. colistrains useful for expression include XL-1, TB1, JM103, BLR, pUC8, pUC9,and pBR329 (Biorad Laboratories, Richmond, Calif.) and pPL and pKK223-3available from (Pharmacia, Piscataway, N.J.).

A bacterial host that expresses a contemplated recombinant E-WE thrombinenzyme is prokaryote, such as E. coli, and a preferred vector includes aprokaryotic replicon; i.e., a DNA sequence having the ability to directautonomous replication and maintenance of the recombinant DNA moleculeextrachromosomally in a prokaryotic host cell transformed therewith.Such replicons are well known in the art. Vectors that include aprokaryotic replicon can also include a prokaryotic promoter regioncapable of directing the expression of a contemplated E-WE thrombin genein a host cell, such as E. coli, transformed therewith.

Promoter sequences compatible with bacterial hosts are typicallyprovided in plasmid vectors containing one or more convenientrestriction sites for insertion of a contemplated DNA segment.Illustratively useful promoters and vectors include the Rec 7 promoterthat is inducible by exogenously supplied nalidixic acid. A morepreferred promoter is present in plasmid vector JHEX25 (Promega,Madison, Wis.) that is inducible by exogenously suppliedisopropyl-β-D-thiogalacto-pyranoside (IPTG). Another preferred promoter,the tac (a hybrid of the trp and lac promoter/operator), is present inplasmid vector pKK223-3 (Pharmacia, Piscataway, N.J.) and is alsoinducible by exogenously supplied IPTG. Further promoters andpromoter/operators include the araB, trp, lac, gal, T7, and the like areuseful in accordance with the instant invention.

The exact details of the expression construct vary according to theparticular host cell that is to be used as well as to the desiredcharacteristics of the expression system, as is well known in the art.For example, for production in S. cerevisiae, the DNA encoding athrombin precursor of the invention is placed into operable linkage witha promoter that is operable in S. cerevisiae and which has the desiredcharacteristics (e.g., inducible/derepressible or constituative), suchas GAL1-10, PHO5, PGK1, GDP1, PMA1, METS, CUP1, GAP, TPI, MF.alpha.1 andMF.alpha.2, as well as the hybrid promoters PGK/.alpha.2, TPI/.alpha.2,GAP/GAL, PGK/GAL, GAP/ADH2, GAP/PHO5, ADH2/PHO5, CYC1/GRE, and PGK/AREand other promoters known in the art.

When other eukaryotic cells are the desired host cell, any promoteractive in the host cell may be utilized. For example, when the desiredhost cell is a mammalian cell line, the promoter can be a viralpromoter/enhancer (e.g., the herpes virus thymidine kinase (TK) promoteror a simian virus promoter (e.g., the SV40 early or late promoter) orthe Adenovirus major late promoter, a long terminal repeat (LTR), suchas the LTR from cytomegalovirus-(CMV), Rous sarcoma virus (RSV) or mousemammary tumor virus (MMTV)) or a mammalian promoter, preferably aninducible promoter such as the metallothionein or glucocorticoidreceptor promoters and the like.

Expression constructs can also include other DNA sequences appropriatefor the intended host cell. For example, expression constructs for usein higher eukaryotic cell lines (e.g., vertebrate and insect cell lines)include a poly-adenylation site and can include an intron (includingsignals for processing the intron), as the presence of an intron appearsto increase mRNA export from the nucleus in many systems. Additionally,a secretion signal sequence operable in the host cell is normallyincluded as part of the construct. The secretion signal sequence can bethe naturally occurring preprothrombin signal sequence, or it can bederived from another gene, such as human serum albumin, humanprothrombin, human tissue plasminogen activator, or preproinsulin. Wherethe expression construct is intended for use in a prokaryotic cell, theexpression construct can include a signal sequence that directstransport of the synthesized polypeptide into the periplasmic space orexpression can be directed intracellularly.

Preferably, the expression construct also comprises a means forselecting for host cells that contain the expression construct (a“selectable marker”). Selectable markers are well known in the art. Forexample, the selectable marker can be a resistance gene, such as aantibiotic resistance gene (e.g., the neo.sup.r gene that confersresistance to the antibiotic gentamycin or the hyg.sup.r gene, thatconfers resistance to the antibiotic hygromycin). Alternatively, theselectable marker can be a gene that complements an auxotrophy of thehost cell. If the host cell is a Chinese hamster ovary (CHO) cell thatlacks the dihydrofolate reductase (dhfr) gene, for example CHO DUXB11cells, a complementing dhfr gene would be preferred.

If the host cell is a yeast cell, the selectable marker is preferably agene that complements an auxotrophy of the cell (for example,complementing genes useful in S. cerevisiae, P. pastoris and S. pombeinclude LEU2, TRP1, TRP1d, URA3, URA3d, HIS3, HIS4, ARG4, LEU2d),although antibiotic resistance markers such as SH BLE, which confersresistance to ZEOCIN®, can also be used. If the host cell is aprokaryotic or higher eukaryotic cell, the selectable marker ispreferably an antibiotic resistance marker (e.g., neo.sup.r).Alternately, a separate selectable marker gene is not included in theexpression vector, and the host cells are screened for the expression ofa thrombin precursor (e.g., upon induction or derepression forcontrollable promoters, or after transfection for a constituitivepromoter, fluorescence-activated cell sorting, FACS, may be used toselect those cells which express the recombinant thrombin precursor).Preferably, the expression construct comprises a separate selectablemarker gene.

A suitable promoter or enhancer, termination sequence and otherfunctionalities for use in the expression of a thrombin precursor ingiven recombinant host cells are well known, as are suitable host cellsfor transfection with nucleic acid encoding the desired variantthrombin. It can be useful to use host cells that are capable ofglycosylating the variant thrombin precursors, which typically includemammalian cells as discussed before.

In addition, host cells are suitable that have been used heretofore toexpress proteolytic enzymes or zymogens in recombinant cell culture, orwhich are known to already express high levels of such enzymes orzymogens in non-recombinant culture. In the latter case, if theendogenous enzyme or thrombin precursor is difficult to separate from avariant thrombin precursor, the endogenous gene should be removed byhomologous recombination or its expression suppressed by cotransfectingthe host cell with nucleic acid encoding an anti-sense sequence that iscomplementary to the RNA encoding the undesired polypeptide. In thiscase, the expression control sequences (e.g., promoter, enhancers, etc.)used by the endogenous expressed gene optimally are used to controlexpression of a thrombin precursor variant.

The following examples are for illustrative purposes and are in no waylimiting.

Example 1 Protocol for E. coli Expression of Thrombin Mutant E-WE

The cDNA corresponding to prethrombin-2 sequence was cloned into pET21avector (Novagen) using the EcoRI and the XhoI restriction sites.Site-directed mutagenesis was carried out using the Quikchange®site-directed mutagenesis kit from Strataqene (La Jolla, Calif.) to makethe thrombin double mutation: W215A/E217A (WE), The E-WEprethrombin-2-encoding vector so prepared was transformed into BL21(DE3) E. coli cells.

The E. coli cells were grown overnight (about 18 hours) in 10 mL of LBmedium with 100 μg/mL ampicillin at 37° C. and 225 rpm. The nextmorning, 3 L of LB medium with 100 μg/mL of ampicillin was inoculatedwith the 10 mL overnight culture. Growth was continued at 37° C. and 225rpm until the cells reached OD₆₀₀=0.6.

Prethrombin-2 expression was initiated by adding IPTG to a finalconcentration of 1 mM. The E. coli cells were cultured for an additional4 hours. The cultures were spun at 3500 rpm for 20 minutes at 4° C.

The supernatant was discarded and the cell paste was frozen at −20° C.The cell paste, from 3 L of LB medium, was thawed at 37° C. andre-suspended in 50 mL of 50 mM Tris pH=7.4 at 25° C., 20 mM EDTA, 1%Triton® X-100, 20 mM DTT. Cells were sonicated on ice for 30 seconds×5(about 1 minute rest) at constant duty, 5½ output, and time-hold. Thewell-homogenized cells were ultra-centrifuged for 30 minutes at 4° C.,30,000 rpm, using a Ti45 rotor.

The supernatant was discarded, and the pellet was re-suspended in 40 mlof 50 mM Tris pH=7.4 at 25° C., 20 mM EDTA, 1M NaCl using gentlevortexing and a spatula. The homogenate was centrifuged for 30 minutes,30,000 rpm, 4° C. Supernatant was discarded, and the pellet wasre-suspended in 40 ml of 50 mM Tris pH=7.4 at 25° C., 20 mM EDTA priorto centrifugation for 30 minutes, 30,000 rpm at 4° C. The supernatantwas discarded, and the pellet was re-suspended in 27 mL of 7 M GdnHCl, 3ml of 0.1% H₂0/TFA, and 30 mM L-cysteine, mixed and allowed to stand at25° C. for 3 hours. The suspension was spun at 30,000 rpm at 4° C. for45 minutes.

Refolding of prethrombin-2 was initiated by flash addition of theunfolded protein into 3 L of refolding buffer, 0.55 M L-arginine, 30%glycerol, 0.2 M NaCl, 1 mM L-cysteine, 0.1% polyoxyethylene 20 cetylether (Brij® 58), 50 mM Tris pH=8 at 25° C. The volume of refoldingbuffer used was such that the final GdnHCl concentration was not morethan 0.15 M. During the addition of the unfolded protein, the refoldingbuffer was stirred at 250 rpm using a magnetic stirrer. After theaddition of unfolded protein, the refolding buffer was left at roomtemperature for 12 hours without stirring.

The solution containing the refolded protein was concentrated fromgenerally 3 L to 500 mL using a Flexstand®, from GE Healthcare(Piscataway, N.J.), with a 10 kDa mwc/1 m² hollow fiber cartridge. Usingthe Flexstand®, the refolded protein was diafiltered against 10 litersof 20 mM Tris pH=8 at 25° C., 50 mM NaCl. Precipitate was removed bycentrifugation and filtration.

Protein solution was loaded onto a 5 mL heparin column, GE Healthcare,at 3 mL/minute. The bound protein was extensively washed by 100 mM NaCl,10 mM Tris pH=8 at 25° C. before elution with a linear gradient of 100mM to 1 M NaCl in 10 mM Tris, pH 8 at 25° C. The elution was monitoredby UV spectroscopy. Prethrombin-2 was activated using a 200 nM finalconcentration of ecarin at 25° C.

To check for the completion of activation, SDS-PAGE was performed on theactivated sample under reducing (by adding 5% v/v of β-mercaptoethanol)and non-reducing conditions.

The activated protein was diluted 4-fold and loaded on the heparincolumn. Bound protein was extensively washed with 100 mM NaCl, 10 mMTris pH=8 at 25° C. before elution with a linear gradient of 100 mM to 1M NaCl in 10 mM Tris, pH 8 at 25° C. Fractions with OD₂₈₀ greater than0.1 were combined, concentrated and applied to detoxi-gel column toremove endotoxins. Concentration of the protein was determined by takingthe OD₂₈₀. The extinction coefficient of recombinant E-WE thrombin at280 nm is 1.83 M⁻¹ cm⁻¹.

The following is a description of the functional characterization ofE-WE thrombin expressed in E. coli. E. coli does not have the capabilityto introduce glycosylation at the sole site in human thrombin atposition N60g. The functional properties of WE thrombin mutant expressedfrom the BHK and from the E. coli expression systems are illustratedbelow in Table 1, with data for wild type (WT) thrombin as a control.The BHK-expressed (mammalian-expressed) WT thrombin was prepared asdiscussed and reported in US Patent Publication 2010/0158890 and inMarino et al., (Jun. 18, 2010) J. Biol. Chem. 285(25):19145-19152.Relevant parameters for all physiological substrates are listed in Table1 below. Solution conditions are 5 mM Tris pH=7.4 at 37° C., 0.1% PEG8000, 145 mM NaCl.

TABLE 1 Catalytic activity (expressed as k_(cat)/K_(m) in mM⁻¹s⁻¹) of WEthrombin mutant expressed in BHK or E. coli Fibrinogen PAR 1 Protein CBHK* 0.89 26 33 E. coli 0.16 10 22 WT* 17,000 26,000 220 *Cantwell andDi Cera, (2000) J. Biol. Chem., 275(51): 39827-39830.

Example 2 In Vivo Studies with E. coli-Expressed Thrombin Mutant E-WE

BHK WE or E. coli E-WE were injected (250 μg/kg, slow bolus, 185 μLvolume) into the femoral vein of C57B16 mice at 250 μg/kg. Blood wasdrawn by cardiac puncture into citrate at 10 minutes, plasma wasprepared by centrifugation, and the plasma activated partialthromboplastin time (APTT) was determined (within 10 minutes of blooddrawing).

The efficacy of the E-WE thrombin was also evaluated in conventionalAPTT assay using the PTT Automate® on Start® 4 instruments (DiagnosticaStago, Asnieres, France). For measurements of APTT (in seconds), fiftyμL aliquots of platelet-poor plasma were transferred to disposablecuvettes (Diagnostica Stago, Parsippany, N.J.), and after addition ofthe APTT reagent and pre-incubation at 37° C., samples were run induplicate. Therapeutic concentrations of DTIs (argatroban 0.5-1 μg/ml;lepirudin 0.1-1 μg/ml; bivalirudin 1-10 μg/ml), and heparin (0.2-0.5U/ml) were used to pre-treat plasma. Effects of a E-WE thrombin mutanton APTT in DTI-treated plasma were evaluated after adding thrombinmutant (5 μg/ml, final concentration). Catalytic-site blocked thrombin(thrombin saturated with Phe-Pro-Arg-chlormethylketone; FPRck) was usedat 100 μg/ml in some studies for comparison.

A plasma aliquot was incubated at a 37° C. for 60 minutes from blooddrawing, and the APTT test was repeated. Decrease in APTT compared tothe 10 minute sample indicates the presence of APC. Blood counts weredetermined to evaluate potential adverse effects (significant plateletconsumption, bleeding).

The data are found in Table 2 below. The conclusion reached is that inmice, E. coli E-WE is a more potent anticoagulant than BHK-expressed WE.

TABLE 2 Evaluation of the anticoagulant activity of high dose BHK vs. E.coli E-WE in vivo, in mouse model of endogenous APC generation 10 min*60 min WT APTT APTT WBC* RBC* HCT* PLT* BHK WE 24.3 38.7 25.4 12.52 9.7742.8 870 22 35.4 14.9 10.16 9.1 40.9 1053 23.2 35.7 18.4 9.52 9.54 43.31040 E. coli 21 47.8 24.4 9.4 9.49 42.4 873 E-WE 20.5 90.9 33.9 3.8 8.3936.7 795 22.3 73.9 29.9 9.64 9.04 40.2 976 24 55.9 28.4 9.94 9.04 40.1927 20.2 55.9 29.9 12.28 9.75 42.9 996 21.4 76.1 29.9 12.9 9.18 40.7 857Control 25 24.4 22.8 7.78 8.97 40.5 1007 *min = minute; red blood cellcount (RBC); white blood cell count (WBC); hematocrit (HCT); andplatelet count (PLT)

Example 3 Comparison of WE Properties on Expression in BHK or E. coli

Table 3, below, presents a summary of the functional properties of WEthrombin prepared from WE prethrombin-2 expressed in BHK and E. colicells. Values are listed for the k_(cat)/K_(m) for the hydrolysis of achromogenic substrate (FPR), and the physiological substrates fibrinogen(FpA), PAR 1 and protein C (PC) activation.

In the case of wild-type thrombin, BHK and E. coli productions areequivalent. In the case of a WE thrombin, BHK produces a construct withsignificantly higher activity toward FPR and fibrinogen. A E-WE thrombinmade in E. coli consistently shows lower specificity toward fibrinogencompared to the BHK-expressed construct resulting in higheranticoagulant activity. This same effect is also seen in vivo.

Included in the Table 3 are data from BHK and E. coli production ofanother anticoagulant thrombin mutant, Δ146-149e. As shown in Table 3,below, there is no difference in anticoagulant activity betweenΔ146-149e constructs expressed in either BHK and E. coli.

An unexpected result happens for a WE thrombin prepared from a thrombinprecursor expressed in E. coli, most likely due to retention of a longerA chain and part of Fragment 2 (incompletely cleaved) in the BHKconstruct due to the different activation. There is an Arg-Thr cleavagesite sequence about 13 residues upstream of the usual Arg-Thr cleavagesite within the Fragment 2 position of the molecule. That unexpectedresult is a markedly lower activity of the E-WE thrombin prepared frombacteria-expressed prethrombin-2 and that expressed in BHK cells.

The table below also contains similar data for E-WE thrombin expressedin E. coli and prepared by activation using only ecarin, whosepreparation is discussed hereinafter.

TABLE 3 Summary of the functional properties of thrombin constructs madein BHK and E. coli Protein C Fibrinogen Par 1 FPR Activation (mM⁻¹s⁻¹)(μM⁻¹s⁻¹) (μM⁻¹s⁻¹) (μM⁻¹s⁻¹) Method E. coli WT 220 ± 1  9.6 ± 0.5 24 ±1  26 ± 1  Ecarin BHK WT 220 ± 1  17 ± 1  26 ± 1  37 ± 2  ProthrombinaseE. coli Δ146-149e 35 ± 1 0.11 ± 0.01 0.13 ± 0.01 1.6 ± 0.1Prothrombinase, Ecarin BHK Δ146-149e 13 ± 1 0.071 ± 0.004 0.12 ± 0.010.26 ± 0.01 Ecarin E. coli E-WE  9.26 ± 0.036 0.000161 ± 1.9e−8   0.010± 1.3e−5  0.0011 ± 1.08e−6 Prothrombinase + (Lot 1) FZ Ecarin E. coliE-WE 25 ± 1 0.00013 ± 0.00001 0.0040 ± 0.0002 0.0015 ± 0.0001Prothrombinase (Lot 2) Ecarin, AC E. coli E-WE 14 ± 1 0.0011 ± 0.00010.096 ± 0.005 0.0023 ± 0.0001 Ecarin, AC (Lot 3) E. coli E-WE 16 ± 10.00019 ± 0.00001 0.032 ± 0.002 0.0012 ± 0.0001 Prothrombinase, (Lot 4)Ecarin, AC E. coli E-WE  3.7 ± 0.07 0.00017 ± 7e−5   0.0094 ± 3.9e−4 0.0016 ± 2.03e−6 Prothrombinase + (Lot 5) Ecarin E. coli E-WE  24.6 ±0.045 0.000184 ± 4.12e−9  0.0085 ± 3.9e−4 0.0035 ± 2.9e−5Prothrombinase + (Lot 6, FZ) Ecarin E. coli E-WE  26.6 ± 0.062 0.00027 ±1.1e−4   0.0099 ± 1.27e−4 0.0014 ± 6e−6  Ecarin Only Two ecarin sitesLot 1 BHK WE, * 33 ± 2 0.00089 ± 7e−5   0.026 ± 1e−3  0.0028 ± 1e−5 Prothrombinase 100 nM TM Only BHK WE  4.1 ± 0.3 0.0052 ± 0.0003 0.017 ±0.001 0.018 ± 0.002 Prothrombinase, Ecarin * Cantwell and Di Cera,(2000) J. Biol. Chem., 275(51): 39827-39830.

The above data were collected under the following protein C reactionconditions: 50 nM thrombomodulin (TM), 50 nM protein C (PC), 0.5 nMthrombin and the following buffer conditions: 145 mM NaCl, 5 mM Tris pH7.4 at 37° C., 0.1% PEG 8000.

The table above summarizes the results of different lots of E-WEthrombin prepared from prethrombin-2 expressed in E. coli cells and WEthrombin prepared from prethrombin-1 expressed in BHK cells, activatedwith prothrombinase and Echis carinatus snake venom prothrombinactivator (ecarin) as the E. coli construct. There is highreproducibility in the different lots activated this way, but a largedifference in fibrinogen cleavage caused by WE thrombin prepared fromprethrombin-1 expressed in BHK cells and E-WE thrombin produced fromprethrombin-2 expressed in E. coli.

There is also a large difference between E-WE thrombin prepared fromprethrombin-2 expressed in E. coli and activated with prothrombinaseplus ecarin vs the use of ecarin and AC venoms.

The specifics of why expression of WE prethrombin-1 in BHK and E-WEprethrombin-2 in E. coli produce different activities of WE thrombineven after the same activation process remain unknown. However, theresults are reproducible and the data from in vivo studies confirm thedata obtained from in vitro studies.

It is noted that wild type thrombin and the anticoagulant mutantΔ146-149e feature the same properties when made from correspondingprethrombin molecules expressed in BHK or E. coli cells by the samecoding sequence. There is therefore something special about E-WEthrombin prepared from E. coli-expressed prethrombin-2 that is activatedwith prothrombinase and ecarin that produces an enhanced anticoagulantprofile.

Example 4 E-WE Thrombin Prepared from an Ecarin Cleavage Site atResidues 325-327

The wild-type prethrombin-2 sequence contains residues FNPRTF (SEQ IDNO:11) at positions 324-329 from the N-terminus of the preprothrombinpolypeptide. That sequence was replaced with the engineered sequenceFDGRTF (SEQ ID NO:12), that placed the ecarin cleavage site—DGR—atpositions 325-327 of the expressed sequence. Only two amino acidresidues needed to be changed. The naturally-occurring cleavage site forecarin is YIDGRIV (SEQ ID NO:13), whose cleavage separates the thrombinA and B chains.

To accomplish these changes, two primers were designed coding for thealtered sequence in both the 5′ to 3′ and reverse complement direction.Using PCR site directed mutagenesis, the mutation was made andultimately confirmed with DNA sequencing. The new plasmid construct fora E-WE thrombin precursor with an additional ecarin site was used toover express the protein in an established E. coli expression system.

Upon refolding and initial purification as discussed above, the E-WEprethrombin-2 was processed using the snake venom derived proteaseecarin. The expected cleavage took place between the A and B chains toform an A chain-extended E-WE thrombin. Cleavage also occurred at theengineered ecarin site that had been introduced preceding the A chain.This cleavage resulted in a properly processed N-terminus as confirmedby N-terminal sequencing of both the A and B chains.

When subjected to standard in vitro biochemical characterization, theE-WE-thrombin produced using this new processing method behaved in anequivalent manner to E-WE-thrombin produced using the original vector(Table 3, above) and activated by prothrombinase complex and ecarin.

The alternative pathway promoted by FXa proceeds through the generationof the inactive precursor prethrombin-2 by cleaving at Arg271 and thenactivation to thrombin by cleaving at Arg320. Another strategy toproduce thrombin from the inactive precursor prothrombin has beendeveloped by using ecarin, a zinc metalloprotease mostly present indifferent snake venoms. Indeed, ecarin has a FXa-similar activity,specifically cleaves the C-terminal of Arg320 and irreversibly activatesprothrombin/prethrombin-2 into meizothrombin/thrombin.

Once activated, meizothrombin/thrombin itself cleaves at Arg284 togenerate the correct A chain, and thus, the mature enzyme. As occurs inthe absence of prothrombinase complex or in the case of a poorly activethrombin mutant, a single ecarin cleavage is not sufficient to generatethe mature and physiologically relevant thrombin enzyme.

Listed Sequences

WE Thrombin SEQ ID NO: 1 TFGSGEADCG LRPLFEKKSL EDKTERELLE SYIDGRIVEGSDAEIGMSPW QVMLFRKSPQ ELLCGASLIS DRWVLTAAHCLLYPPWDKNF TENDLLVRIG KHSRTRYERN IEKISMLEKIYIHPRYNWRE NLDRDIALMK LKKPVAFSDY IHPVCLPDRETAASLLQAGY KGRVTGWGNL KETWTANVGK GQPSVLQVVNLPIVERPVCK DSTRIRITDN MFCAGYKPDE GKRGDACEGDSGGPFVMKSP FNNRWYQMGI VSAGAGCDRD GKYGFYTHVF RLKKWIQKVI DQFGE ThrombinSEQ ID NO: 2 TFGSGEADCG LRPLFEKKSL EDKTERELLE SYIDGRIVEGSDAEIGMSPW QVMLFRKSPQ ELLCGASLIS DRWVLTAAHCLLYPPWDKNF TENDLLVRIG KHSRTRYERN IEKISMLEKIYIHPRYNWRE NLDRDIALMK LKKPVAFSDY IHPVCLPDRETAASLLQAGY KGRVTGWGNL KETWTANVGK GQPSVLQVVNLPIVERPVCK DSTRIRITDN MFCAGYKPDE GKRGDACEGDSGGPFVMKSP FNNRWYQMGI VSWGEGCDRD GKYGFYTHVF RLKKWIQKVI DQFGEPreprothrombin SEQ ID NO: 3 MAHVRGLQLP GCLALAALCS LVHSQHVFLA PQQARSLLQRVRRANTFLEE VRKGNLEREC VEETCSYEEA FEALESSTATDVFWAKYTAC ETARTPRDKL AACLEGNCAE GLGTNYRGHVNITRSGIECQ LWRSRYPHKP EINSTTHPGA DLQENFCRNPDSSTTGPWCY TTDPTVRRQE CSIPVCGQDQ VTVAMTPRSEGSSVNLSPPL EQCVPDRGQQ YQGRLAVTTH GLPCLAWASAQAKALSKHQD FNSAVQLVEN FCRNPDGDEE GVWCYVAGKPGDFGYCDLNY CEEAVEEETG DGLDEDSDRA IEGRTATSEYQTFFNPRTFG SGEADCGLRP LFEKKSLEDK TERELLESYIDGRIVEGSDA EIGMSPWQVM LFRKSPQELL CGASLISDRWVLTAAHCLLY PPWDKNFTEN DLLVRIGKHS RTRYERNIEKISMLEKIYIH PRYNWRENLD RDIALMKLKK PVAFSDYIHPVCLPDRETAA SLLQAGYKGR VTGWGNLKET WTANVGKGQPSVLQVVNLPI VERPVCKDST RIRITDNMFC AGYKPDEGKRGDACEGDSGG PFVMKSPFNN RWYQMGIVSW GEGCDRDGKY GFYTHVFRLK KWIQKVIDQF GEEcarin-activatable E-WE Preprothrombin SEQ ID NO: 4MAHVRGLQLP GCLALAALCS LVHSQHVFLA PQQARSLLQRVRRANTFLEE VRKGNLEREC VEETCSYEEA FEALESSTATDVFWAKYTAC ETARTPRDKL AACLEGNCAE GLGTNYRGHVNITRSGIECQ LWRSRYPHKP EINSTTHPGA DLQENFCRNPDSSTTGPWCY TTDPTVRRQE CSIPVCGQDQ VTVAMTPRSEGSSVNLSPPL EQCVPDRGQQ YQGRLAVTTH GLPCLAWASAQAKALSKHQD FNSAVQLVEN FCRNPDGDEE GVWCYVAGKPGDFGYCDLNY CEEAVEEETG DGLDEDSDRA IEGRTATSEYQTFFDGRTFG SGEADCGLRP LFEKKSLEDK TERELLESYIDGRIVEGSDA EIGMSPWQVM LFRKSPQELL CGASLISDRWVLTAAHCLLY PPWDKNFTEN DLLVRIGKHS RTRYERNIEKISMLEKIYIH PRYNWRENLD RDIALMKLKK PVAFSDYIHPVCLPDRETAA SLLQAGYKGR VTGWGNLKET WTANVGKGQPSVLQVVNLPI VERPVCKDST RIRITDNMFC AGYKPDEGKRGDACEGDSGG PFVMKSPFNN RWYQMGIVSA GAGCDRDGKY GFYTHVFRLK KWIQKVIDQF GEEcarin-activatable E-WE Prethrombin-2 SEQ ID NO: 5TATSEYQTFF DGRTFGSGEA DCGLRPLFEK KSLEDKTERELLESYIDGRI VEGSDAEIGM SPWQVMLFRK SPQELLCGASLISDRWVLTA AHCLLYPPWD KNFTENDLLV RIGKHSRTRYERNIEKISML EKIYIHPRYN WRENLDRDIA LMKLKKPVAFSDYIHPVCLP DRETAASLLQ AGYKGRVTGW GNLKETWTANVGKGQPSVLQ VVNLPIVERP VCKDSTRIRI TDNMFCAGYKPDEGKRGDAC EGDSGGPFVM KSPFNNRWYQ MGIVSAGAGCDRDGKYGFYT HVFRLKKWIQ KVIDQFGE Ecarin-activatable PreprothrombinSEQ ID NO: 6 MAHVRGLQLP GCLALAALCS LVHSQHVFLA PQQARSLLQRVRRANTFLEE VRKGNLEREC VEETCSYEEA FEALESSTATDVFWAKYTAC ETARTPRDKL AACLEGNCAE GLGTNYRGHVNITRSGIECQ LWRSRYPHKP EINSTTHPGA DLQENFCRNPDSSTTGPWCY TTDPTVRRQE CSIPVCGQDQ VTVAMTPRSEGSSVNLSPPL EQCVPDRGQQ YQGRLAVTTH GLPCLAWASAQAKALSKHQD FNSAVQLVEN FCRNPDGDEE GVWCYVAGKPGDFGYCDLNY CEEAVEEETG DGLDEDSDRA IEGRTATSEYQTFFDGRTFG SGEADCGLRP LFEKKSLEDK TERELLESYIDGRIVEGSDA EIGMSPWQVM LFRKSPQELL CGASLISDRWVLTAAHCLLY PPWDKNFTEN DLLVRIGKHS RTRYERNIEKISMLEKIYIH PRYNWRENLD RDIALMKLKK PVAFSDYIHPVCLPDRETAA SLLQAGYKGR VTGWGNLKET WTANVGKGQPSVLQVVNLPI VERPVCKDST RIRITDNMFC AGYKPDEGKRGDACEGDSGG PFVMKSPFNN RWYQMGIVSW GEGCDRDGKY GFYTHVFRLK KWIQKVIDQF GEEcarin-activatable Prethrombin-2 SEQ ID NO: 7TATSEYQTFF DGRTFGSGEA DCGLRPLFEK KSLEDKTERELLESYIDGRI VEGSDAEIGM SPWQVMLFRK SPQELLCGASLISDRWVLTA AHCLLYPPWD KNFTENDLLV RIGKHSRTRYERNIEKISML EKIYIHPRYN WRENLDRDIA LMKLKKPVAFSDYIHPVCLP DRETAASLLQ AGYKGRVTGW GNLKETWTANVGKGQPSVLQ VVNLPIVERP VCKDSTRIRI TDNMFCAGYKPDEGKRGDAC EGDSGGPFVM KSPFNNRWYQ MGIVSWGEGCDRDGKYGFYT HVFRLKKWIQ KVIDQFGEEcarin-activatable Δ146-149e Prethrombin-2 SEQ ID NO: 8TATSEYQTFF DGRTFGSGEA DCGLRPLFEK KSLEDKTERELLESYIDGRI VEGSDAEIGM SPWQVMLFRK SPQELLCGASLISDRWVLTA AHCLLYPPWD KNFTENDLLV RIGKHSRTRYERNIEKISML EKIYIHPRYN WRENLDRDIA LMKLKKPVAFSDYIHPVCLP DRETAASLLQ AGYKGRVTGW GNLKGKGQPSVLQVVNLPIV ERPVCKDSTR IRITDNMFCA GYKPDEGKRGDACEGDSGGP FVMKSPFNNR WYQMGIVSWG EGCDRDGKYG FYTHVFRLKK WIQKVIDQFG EWE Preprothrombin SEQ ID NO: 9MAHVRGLQLP GCLALAALCS LVHSQHVFLA PQQARSLLQRVRRANTFLEE VRKGNLEREC VEETCSYEEA FEALESSTATDVFWAKYTAC ETARTPRDKL AACLEGNCAE GLGTNYRGHVNITRSGIECQ LWRSRYPHKP EINSTTHPGA DLQENFCRNPDSSTTGPWCY TTDPTVRRQE CSIPVCGQDQ VTVAMTPRSEGSSVNLSPPL EQCVPDRGQQ YQGRLAVTTH GLPCLAWASAQAKALSKHQD FNSAVQLVEN FCRNPDGDEE GVWCYVAGKPGDFGYCDLNY CEEAVEEETG DGLDEDSDRA IEGRTATSEYQTFFNPRTFG SGEADCGLRP LFEKKSLEDK TERELLESYIDGRIVEGSDA EIGMSPWQVM LFRKSPQELL CGASLISDRWVLTAAHCLLY PPWDKNFTEN DLLVRIGKHS RTRYERNIEKISMLEKIYIH PRYNWRENLD RDIALMKLKK PVAFSDYIHPVCLPDRETAA SLLQAGYKGR VTGWGNLKET WTANVGKGQPSVLQVVNLPI VERPVCKDST RIRITDNMFC AGYKPDEGKRGDACEGDSGG PFVMKSPFNN RWYQMGIVSA GAGCDRDGKY GFYTHVFRLK KWIQKVIDQF GEWE Prethrombin-2 SEQ ID NO: 10TATSEYQTFF NPRTFGSGEA DCGLRPLFEK KSLEDKTERELLESYIDGRI VEGSDAEIGM SPWQVMLFRK SPQELLCGASLISDRWVLTA AHCLLYPPWD KNFTENDLLV RIGKHSRTRYERNIEKISML EKIYIHPRYN WRENLDRDIA LMKLKKPVAFSDYIHPVCLP DRETAASLLQ AGYKGRVTGW GNLKETWTANVGKGQPSVLQ VVNLPIVERP VCKDSTRIRI TDNMFCAGYKPDEGKRGDAC EGDSGGPFVM KSPFNNRWYQ MGIVSAGAGCDRDGKYGFYT HVFRLKKWIQ KVIDQFGE SEQ ID NO: 11 FNPRTF SEQ ID NO: 12 FDGRTFSEQ ID NO: 13 YIDGRIV Ecarin-activatable E-WE Thrombin Precursor ASEQ ID NO: 14 DGRTFGSGEA DCGLRPLFEK KSLEDKTERE LLESYIDGRIVEGSDAEIGM SPWQVMLFRK SPQELLCGAS LISDRWVLTAAHCLLYPPWD KNFTENDLLV RIGKHSRTRY ERNIEKISMLEKIYIHPRYN WRENLDRDIA LMKLKKPVAF SDYIHPVCLPDRETAASLLQ AGYKGRVTGW GNLKETWTAN VGKGQPSVLQVVNLPIVERP VCKDSTRIRI TDNMFCAGYK PDEGKRGDACEGDSGGPFVM KSPFNNRWYQ MGIVSAGAGC DRDGKYGFYT HVFRLKKWIQ KVIDQFGESEQ ID NO: 15 EGRTFGSGEA DCGLRPLFEK KSLEDKTERE LLESYIDGRIVEGSDAEIGM SPWQVMLFRK SPQELLCGAS LISDRWVLTAAHCLLYPPWD KNFTENDLLV RIGKHSRTRY ERNIEKISMLEKIYIHPRYN WRENLDRDIA LMKLKKPVAF SDYIHPVCLPDRETAASLLQ AGYKGRVTGW GNLKETWTAN VGKGQPSVLQVVNLPIVERP VCKDSTRIRI TDNMFCAGYK PDEGKRGDACEGDSGGPFVM KSPFNNRWYQ MGIVSAGAGC DRDGKYGFYT HVFRLKKWIQ KVIDQFGEEcarin-activatable Thrombin Precursor A SEQ ID NO: 16DGRTFGSGEA DCGLRPLFEK KSLEDKTERE LLESYIDGRIVEGSDAEIGM SPWQVMLFRK SPQELLCGAS LISDRWVLTAAHCLLYPPWD KNFTENDLLV RIGKHSRTRY ERNIEKISMLEKIYIHPRYN WRENLDRDIA LMKLKKPVAF SDYIHPVCLPDRETAASLLQ AGYKGRVTGW GNLKETWTAN VGKGQPSVLQVVNLPIVERP VCKDSTRIRI TDNMFCAGYK PDEGKRGDACEGDSGGPFVM KSPFNNRWYQ MGIVSWGEGC DRDGKYGFYT HVFRLKKWIQ KVIDQFGESEQ ID NO: 17 EGRTFGSGEA DCGLRPLFEK KSLEDKTERE LLESYIDGRIVEGSDAEIGM SPWQVMLFRK SPQELLCGAS LISDRWVLTAAHCLLYPPWD KNFTENDLLV RIGKHSRTRY ERNIEKISMLEKIYIHPRYN WRENLDRDIA LMKLKKPVAF SDYIHPVCLPDRETAASLLQ AGYKGRVTGW GNLKETWTAN VGKGQPSVLQVVNLPIVERP VCKDSTRIRI TDNMFCAGYK PDEGKRGDACEGDSGGPFVM KSPFNNRWYQ MGIVSWGEGC DRDGKYGFYT HVFRLKKWIQ KVIDQFGEWild Type Prothrombin SEQ ID NO: 18ANTFLEEVRK GNLERECVEE TCSYEEAFEA LESSTATDVFWAKYTACETA RTPRDKLAAC LEGNCAEGLG TNYRGHVNITRSGIECQLWR SRYPHKPEIN STTHPGADLQ ENFCRNPDSSTTGPWCYTTD PTVRRQECSI PVCGQDQVTV AMTPRSEGSSVNLSPPLEQC VPDRGQQYQG RLAVTTHGLP CLAWASAQAKALSKHQDFNS AVQLVENFCR NPDGDEEGVW CYVAGKPGDFGYCDLNYCEE AVEEETGDGL DEDSDRAIEG RTATSEYQTFFNPRTFGSGE ADCGLRPLFE KKSLEDKTER ELLESYIDGRIVEGSDAEIG MSPWQVMLFR KSPQELLCGA SLISDRWVLTAAHCLLYPPW DKNFTENDLL VRIGKHSRTR YERNIEKISMLEKIYIHPRY NWRENLDRDI ALMKLKKPVA FSDYIHPVCLPDRETAASLL QAGYKGRVTG WGNLKETWTA NVGKGQPSVLQVVNLPIVER PVCKDSTRIR ITDNMFCAGY KPDEGKRGDACEGDSGGPFV MKSPFNNRWY QMGIVSWGEG CDRDGKYGFY THVFRLKKWI QKVIDQFGEWE Prothrombin SEQ ID NO: 19 ANTFLEEVRK GNLERECVEE TCSYEEAFEA LESSTATDVFWAKYTACETA RTPRDKLAAC LEGNCAEGLG TNYRGHVNITRSGIECQLWR SRYPHKPEIN STTHPGADLQ ENFCRNPDSSTTGPWCYTTD PTVRRQECSI PVCGQDQVTV AMTPRSEGSSVNLSPPLEQC VPDRGQQYQG RLAVTTHGLP CLAWASAQAKALSKHQDFNS AVQLVENFCR NPDGDEEGVW CYVAGKPGDFGYCDLNYCEE AVEEETGDGL DEDSDRAIEG RTATSEYQTFFNPRTFGSGE ADCGLRPLFE KKSLEDKTER ELLESYIDGRIVEGSDAEIG MSPWQVMLFR KSPQELLCGA SLISDRWVLTAAHCLLYPPW DKNFTENDLL VRIGKHSRTR YERNIEKISMLEKIYIHPRY NWRENLDRDI ALMKLKKPVA FSDYIHPVCLPDRETAASLL QAGYKGRVTG WGNLKETWTA NVGKGQPSVLQVVNLPIVER PVCKDSTRIR ITDNMFCAGY KPDEGKRGDACEGDSGGPFV MKSPFNNRWY QMGIVSAGAG CDRDGKYGFY THVFRLKKWI QKVIDQFGEEcarin-activatable E-WE Prothrombin SEQ ID NO: 20ANTFLEEVRK GNLERECVEE TCSYEEAFEA LESSTATDVFWAKYTACETA RTPRDKLAAC LEGNCAEGLG TNYRGHVNITRSGIECQLWR SRYPHKPEIN STTHPGADLQ ENFCRNPDSSTTGPWCYTTD PTVRRQECSI PVCGQDQVTV AMTPRSEGSSVNLSPPLEQC VPDRGQQYQG RLAVTTHGLP CLAWASAQAKALSKHQDFNS AVQLVENFCR NPDGDEEGVW CYVAGKPGDFGYCDLNYCEE AVEEETGDGL DEDSDRAIEG RTATSEYQTFFDGRTFGSGE ADCGLRPLFE KKSLEDKTER ELLESYIDGRIVEGSDAEIG MSPWQVMLFR KSPQELLCGA SLISDRWVLTAAHCLLYPPW DKNFTENDLL VRIGKHSRTR YERNIEKISMLEKIYIHPRY NWRENLDRDI ALMKLKKPVA FSDYIHPVCLPDRETAASLL QAGYKGRVTG WGNLKETWTA NVGKGQPSVLQVVNLPIVER PVCKDSTRIR ITDNMFCAGY KPDEGKRGDACEGDSGGPFV MKSPFNNRWY QMGIVSAGAG CDRDGKYGFY THVFRLKKWI QKVIDQFGESEQ ID NO: 21 ANTFLEEVRK GNLERECVEE TCSYEEAFEA LESSTATDVFWAKYTACETA RTPRDKLAAC LEGNCAEGLG TNYRGHVNITRSGIECQLWR SRYPHKPEIN STTHPGADLQ ENFCRNPDSSTTGPWCYTTD PTVRRQECSI PVCGQDQVTV AMTPRSEGSSVNLSPPLEQC VPDRGQQYQG RLAVTTHGLP CLAWASAQAKALSKHQDFNS AVQLVENFCR NPDGDEEGVW CYVAGKPGDFGYCDLNYCEE AVEEETGDGL DEDSDRAIEG RTATSEYQTFFEGRTFGSGE ADCGLRPLFE KKSLEDKTER ELLESYIDGRIVEGSDAEIG MSPWQVMLFR KSPQELLCGA SLISDRWVLTAAHCLLYPPW DKNFTENDLL VRIGKHSRTR YERNIEKISMLEKIYIHPRY NWRENLDRDI ALMKLKKPVA FSDYIHPVCLPDRETAASLL QAGYKGRVTG WGNLKETWTA NVGKGQPSVLQVVNLPIVER PVCKDSTRIR ITDNMFCAGY KPDEGKRGDACEGDSGGPFV MKSPFNNRWY QMGIVSAGAG CDRDGKYGFY THVFRLKKWI QKVIDQFGEE-WE Thrombin with Ecarin Site SEQ ID NO: 22 DGRTFGSGE ADCGLRPLFE KKSLEDKTER ELLESYIDGRIVEGSDAEIG MSPWQVMLFR KSPQELLCGA SLISDRWVLTAAHCLLYPPW DKNFTENDLL VRIGKHSRTR YERNIEKISMLEKIYIHPRY NWRENLDRDI ALMKLKKPVA FSDYIHPVCLPDRETAASLL QAGYKGRVTG WGNLKETWTA NVGKGQPSVLQVVNLPIVER PVCKDSTRIR ITDNMFCAGY KPDEGKRGDACEGDSGGPFV MKSPFNNRWY QMGIVSAGAG CDRDGKYGFY THVFRLKKWI QKVIDQFGESEQ ID NO: 23  EGRTFGSGE ADCGLRPLFE KKSLEDKTER ELLESYIDGRIVEGSDAEIG MSPWQVMLFR KSPQELLCGA SLISDRWVLTAAHCLLYPPW DKNFTENDLL VRIGKHSRTR YERNIEKISMLEKIYIHPRY NWRENLDRDI ALMKLKKPVA FSDYIHPVCLPDRETAASLL QAGYKGRVTG WGNLKETWTA NVGKGQPSVLQVVNLPIVER PVCKDSTRIR ITDNMFCAGY KPDEGKRGDACEGDSGGPFV MKSPFNNRWY QMGIVSAGAG CDRDGKYGFY THVFRLKKWI QKVIDQFGEWild Type Thrombin with Ecarin Site SEQ ID NO: 24 DGRTFGSGE ADCGLRPLFE KKSLEDKTER ELLESYIDGRIVEGSDAEIG MSPWQVMLFR KSPQELLCGA SLISDRWVLTAAHCLLYPPW DKNFTENDLL VRIGKHSRTR YERNIEKISMLEKIYIHPRY NWRENLDRDI ALMKLKKPVA FSDYIHPVCLPDRETAASLL QAGYKGRVTG WGNLKETWTA NVGKGQPSVLQVVNLPIVER PVCKDSTRIR ITDNMFCAGY KPDEGKRGDACEGDSGGPFV MKSPFNNRWY QMGIVSWGEG CDRDGKYGFY THVFRLKKWI QKVIDQFGESEQ ID NO: 25  EGRTFGSGE ADCGLRPLFE KKSLEDKTER ELLESYIDGRIVEGSDAEIG MSPWQVMLFR KSPQELLCGA SLISDRWVLTAAHCLLYPPW DKNFTENDLL VRIGKHSRTR YERNIEKISMLEKIYIHPRY NWRENLDRDI ALMKLKKPVA FSDYIHPVCLPDRETAASLL QAGYKGRVTG WGNLKETWTA NVGKGQPSVLQVVNLPIVER PVCKDSTRIR ITDNMFCAGY KPDEGKRGDACEGDSGGPFV MKSPFNNRWY QMGIVSWGEG CDRDGKYGFY THVFRLKKWI QKVIDQFGE

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) is to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventor for carrying out the invention.Variations of those preferred embodiments can become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventor expects skilled artisans to employ such variations asappropriate, and the inventor intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

Because many possible embodiments can be made of the invention withoutdeparting from the scope thereof, it is to be understood that all matterherein set forth is to be interpreted as illustrative, and not in alimiting sense.

1. A polypeptide that is a recombinant thrombin precursor comprising thesequence Asp/Glu-Gly-Arg peptide-bonded to the incipient N-terminalresidue of the thrombin A chain.
 2. The polypeptide according to claim 1having an amino acid residue sequence of up to about 622 residues. 3.The polypeptide according to claim 1, wherein said Asp/Glu-Gly-Argsequence is present 298-296 amino acid residues from thecarboxy-terminus.
 4. The polypeptide according to claim 1, wherein saidthrombin precursor, except for said Asp/Glu-Gly-Arg sequence, containsthe amino acid residue sequence of wild type human thrombin of SEQ IDNO:2.
 5. The polypeptide according to claim 4, wherein said thrombinprecursor is that of sequence of SEQ ID NOs:6 or
 7. 6. The polypeptideaccording to claim 1, wherein said thrombin precursor contains an aminoacid residue sequence that is at least 95 percent identical to the aminoacid residue sequence of wild type human thrombin of SEQ ID NO:2.
 7. Thepolypeptide according to claim 6, wherein said thrombin precursor isthat of sequence of SEQ ID NOs:4, 5, or
 8. 8. The polypeptide accordingto claim 6, wherein said thrombin precursor includes the amino acidsequence of SEQ ID NOs:1, 9 or
 10. 9. The polypeptide according to claim1, wherein said thrombin precursor is expressed in eukaryotic cells. 10.The polypeptide according to claim 9, wherein said eukaryotic cells aremammalian cells.
 11. The polypeptide according to claim 1 that is afusion polypeptide whose N-terminal portion is a convenientexpression/purification sequence, whose C-terminal portion is a desiredthrombin sequence, and wherein said two portions are joined by the aminoacid residues of an ecarin cleavage site.
 12. The polypeptide accordingto claim 1, wherein said thrombin precursor is expressed in prokaryoticcells.
 13. The polypeptide according to claim 12, wherein saidprokaryotic cells are bacteria cells.
 14. The polypeptide according toclaim 13, wherein said bacteria cells are E. coli cells.
 15. Abacteria-expressed E-WE thrombin precursor having an amino acid residuesequence of up to 622 residues that includes the amino acid residuesequence of SEQ ID NO:1.
 16. The bacteria-expressed E-WE thrombinprecursor according to claim 15 that is E-WE preprothrombin of SEQ IDNO:9.
 17. The bacteria-expressed E-WE thrombin precursor according toclaim 15 that is E-WE prethrombin-2 of SEQ ID NO:
 10. 18. Thebacteria-expressed E-WE thrombin precursor according to claim 15,wherein the bacteria is E. coli.
 19. A kit for the preparation ofthrombin, said kit comprising a package containing a recombinantthrombin precursor of claim 1 in an amount sufficient for at least oneuse and instructions for thrombin preparation using said thrombinprecursor.
 20. The kit according to claim 19, further including a secondpackage containing an effective amount of ecarin.
 21. The kit accordingto claim 19, wherein said thrombin precursor, except for saidAsp/Glu-Gly-Arg sequence, contains the amino acid residue sequence ofwild type human thrombin of SEQ ID NO:2.
 22. The kit according to claim21, wherein said thrombin precursor is that of sequence of SEQ ID NOs:6or
 7. 23. The kit according to claim 19, wherein said thrombin precursorcontains an amino acid residue sequence that is at least 95 percentidentical to the amino acid residue sequence of wild type human thrombinof SEQ ID NO:2.
 24. The kit according to claim 23, wherein said thrombinprecursor is that of sequence of SEQ ID NOs:4, 5, or
 8. 25. The kitaccording to claim 19, wherein said thrombin precursor includes theamino acid sequence of SEQ ID NO:1, 9 or
 10. 26. Bacteria-expressed E-WEthrombin.
 27. The bacteria-expressed E-WE thrombin according to claim26, wherein said bacteria is E. coli.
 28. A pharmaceutical compositionthat comprises an antithrombotic effective amount of bacteria-expressedE-WE thrombin according to claim 26 dissolved or dispersed in apharmaceutically acceptable carrier.
 29. The pharmaceutical compositionaccording to claim 28 that is adapted for parenteral administration. 30.The pharmaceutical composition according to claim 28, wherein saidbacteria-expressed E-WE thrombin is prepared from a fusion polypeptidewhose N-terminal portion is a convenient expression/purificationsequence, whose C-terminal portion is the E-WE thrombin sequence, andwherein said two portions are joined by the amino acid residues of anecarin cleavage site.
 31. A method of treating and preventing thrombosisin a mammal in need thereof that comprises administering anantithrombotic composition according to claim 29 to said mammal.
 32. Themethod according to claim 30, wherein said composition is administeredto a mammal at risk of or having intravascular thrombosis.
 33. Themethod according to claim 32, wherein said administration is repeated aplurality of times.